3-PuGLYCERATE PHOSPHATASE
1N LEAVES

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
DOUGLAS DEY RANDALL
1970

  
   
    

  

LIBRARY

Michigan 3:2 I:
University

This is to certify that the

thesis entitled
5-P-Glycerate PhosPhatase in Leaves
presented by
Douglas Dey Randall g
has been accepted towards fulfillment

of the requirements for

Ph.D. degree in Biochemistry

“NEW

Major professor

Date W 7.!970
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ABSTRACT
3-P~GIJCERATE PHOSPHATASE IN LEAVES
by Douglas Dey Randall

A BAP-glycerate phosphatase was purified from sugar—
cane (Saocharium officiarum) leaves by a sequence of pH
fractionation. acetone and ammonium sulfate precipitations,
and chromatography on G-200 Sephadex, phoSphocellulose and
carboxymethyl Sephadex. The purest preparations were 2530-
fold enriched with 4.h% recovery and had a Specific activity
of 7%. The phosphatase hydrolyzed stoichiometrically 9.3.?-
glycerate to_y-glycerate and inorganic phOSphate. Between
pH #.5 to 7.0 the phoephatase was stable at 4° and -18° and
at room temperatures for brief periods. It was irreversibly
inactivated at alkaline pH.

The activity of the purified enzyme was optimal between
pH 5.7 and 6.0, but in crude leaf extracts the optimum was at
pH 6.3. No requirement for any cofactor was detected. The
Michaelis plots for 3-P-glycerate phoephatase activity were
hyperbolic. The apparent Michaelis constant for 3-P-g1yoerate
‘was 0.28 mM. The temperature optimum was about #2° and the
enZyme was 50% inactivated in 3 minutes at 50°. The isoelec-
’tric point was at pH 6.8.

. The enzyme was most active toward 3-P-glycerate. Phos-
" éphhenolpyruvate was the only physiological substrate which
“She hydrolyzed at a rate greater than half the rate for

Vi

Douglas Dey Randall
BAP-glycerate. p—Nitrophenylphosphate was hydrolyzed at 0.66
of the rate with BAP-glycerate. 0f #1 other phosphate esters,
none were hydrolyzed at rates more than 0.50 that for 3-P-
glycerate. The nucleotidase activity was low. No diester—
ase activity and limited pyrophosphatase activity were
observed. The enzyme did not hydrolyze the C-P bond of phos-
phonic acid derivatives. The purified enzyme was believed
to be free of other phosphatases, since the relative speci-
ficity did not change during the last four steps ofpurifica-
tion. Only one phosphatase was detected by isoeleotric
focusing, cation exchange columns. and kinetic analysis.

The onlyme activity was not affected by extended
dialysis against buffers or 1 mm EDTA. passage through 6-25
Sephadex or treatment with nine metal complexing agents.

Host divalent cations were partially inhibitory. ZnT+. Cu++.
Pb¢+ and sg++ at i uh inhibited the phosphatase greater than
50%. na+, KT. NH¢+ were without effect. The 3-P-g1ycerate

   
 
 
 
 
   
  
  

 

phosphatase was inhibited by typical phOSphatase inhibitors,
fluoride. molybdate, L(+)tartrate and arsenate. Iodoacetate,
arsensite. and p-ohloromecuribenzoate inhibited 13$. 17%. and
2hfl. or the large number of metabolites. analogs or related
compounds which were examined as possible effectors. dihyd-
roxytartrate. (aaino-oxy)acetate. NaBo3. Nazco3. NaHCOB. and
Na303 partially inhibited. L—aaAIanine and L-asparate at

1 In stimulated the activity about 105. Glycidol phosphate
was not hydrolyzed and was an irreversible competitive
inhibitor. Three derivatives of phosphonic acid stimulated

Douglas Dey Randall
L the enzymatic hydrolysis of 3-P-glycerate by 10%.

From gel filtration and sucrose density oentrifugation,
the molecular weight of the enzyme was estimated to be in
the range of 160.000 to 200.000. 0n sucrose density grad—
ients aggregation of the protein occurred at low ionic
; strength. but with 0.25 M KCl in the sucrose one form of the
enzyme was obtained. Two polypeptide bands were observed on
SDS—polyacrylamide electrOphoresis. The molecular weight of
E the major band was about 51.000 and the minor band 62,000.
The molar ratio of the two peptide bands was 3.621.

3-P-Glycerate was found in the mesophyll cells of
sugarcane leaves. Non-aqueous isolation and density centri-
fugation procedures indicated the phOSphatase was in the
cytosol. It was not found in isolated peroxisomes, chloro-
plasts or mitochondria. In etiolated tissue there was 1/10
the ph08phatase activity on a weight basis and 1/# the activ-

    
  
  
  
 
 
 
 
 
 
 

 

ity on a protein basis. Upon exposure to light. the increase
in phosphatase activity paralleled the greening of the tissue,
such that in #7 hours the activity was 87% that of normal
green leaves. The 3aP-glycerate phoSphatase activity exhib-
ited a diurnal variation. During days of high sunlight it
increased 50% and 60% on a protein and chlorophyll basis,
reapectively. late in the afternoon and early darkness.
The amounts of 3-P-g1ycerate phOSphatase and P-glyco-
~1ate phosphatase were determined in to plants. Non-C02-
_ yphotcrespiring plants with the 64-dicarboxylic acid cycle.
._&§enarally had more 3-P-glycerate phOSphatase than P-glyoolate

 

 

  

Douglas Dey Randall

phosphatase. The opposite was the case for C02-photoreSplr-
ing higher plants or C3-pathway plants. The C3-p1ants how—
ever. had sufficient 3-P—glycerate phoSphatase to account for
glycerate formation. Algae were typical of C3-pathway plants,
as were most trees and aquatic plants. The non-vascular
Liverwort, Marchantia, had more 3-P-glycerate phoSphatase
than P-glycolate phosphatase.

The amounts of 3-P-glyoerate and P-glycolate phOSpha-
tases were determined in 15 soybean varieties (Glycine max.
L. Merrill) with known net C02 fixation rates. The amount of
P-glycolate phOSphatase correlated positively with increasing
002 fixation rates; the amount of the 3-P-glycerate phoSpha-
tase correlated inversely with C02 fixation. Assays for
these enzymes may be useful as an index of photosynthesis
potential.

About 30% of the 3-P-glycerate phoSphatase activity in
spinach leaves was bound to the "starch particles" pelleted
through sucrose density gradients. The particulate 3-P-gly-
cerate phoSphatase activity had a specific activity 46—fold
greater than the activity of crude spinach extracts. The
starch particles were so designated because they stain with
KI-Iz, their density was greater than 2.5 M sucrose, and the
phosphatase and reducing sugars were released upon incubation

‘with B-amylase. The phOSphatase could also be released from
" .the particles by 0.35 M Mg012 or extend sonification. Activ-
'.gty was not solubilized by changes in pH, dialysis with or

  

 

Douglas Day Randall
through French pressure cell or homogenization. The 3-P-
glycerate phosphatase solubilized from the particles was
further purified 384-fold to a specific activity of 10.8
with a 3&5 recovery. The pH optimum,the absence of cofactors
and the enzyme kinetics of the particulate enzyme were
similar to the properties of the soluble phosphatase from
sugarcane. A possible role for this phosphatase in regula-
tion of starch formation or degradation is discussed.

Functions of the soluble 3-P-g1ycerate phosphatase
from sugarcane leaves are unknown, but some possibilities
were considered. The phosphatase may be involved in (a)
regulation of the photosynthetic carbon pathways, (b) serine,
glycine and 01 synthesis as an alternate to or in addition
.to the peroxisomal—glycolate pathway. (c) starch synthesis,
or (d) carbon transport between mesophyll and parenchyma
sheath cells.

3-P-GLYCE2ATE PHOSPHATASE IN LEAVE

By

Douglas Day Randall

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

1130108 01" PEIIDSOPH!

Department of Biochemistry

1970

 

 

 

 

G- bee/73
x—n’“ 7/

Dedicated to

My wife. Shirley

and our children. Troy and Reyna

11

 

  

ACKNOWLEDGMENTS

I would like to thank Professor N. E. Talbert for
his enthusiasm,advice and counsel during my research and
graduate training. I also thank Professor Tolbert for his
invaluable contribution towards my development as a bio-
chemist.

My thanks to Dr. Robert Barr and Dr. Robert
Kieckhefer for introducing me to research, biochemistry and
entomology and for the encouragement to enter graduate
study.

0 For their superior technical assistance, my thanks
to Fraulein Angelika Oeser. Mrs. Sandra Wardell and Dennis
Gremel.

My thanks to Professor R. H. Hageman for his person—
al and professional counsel. The cooperation of Professor
R. H. Hageman, and the personnel and the facilities of the
USDA Regional Soybean Laboratory at the University of
Illinois. Urbana for the investigations on the soybean
varieties are gratefully acknowledged.

I appreciate the advice and assistance of my thesis
committee members. Dre. J. L. Fairley, Phil Filner. Cliff
Pollard and John Wilson. My thanks also to various faculty.
staff and students who have contributed immeasurably by
their friendship and interest. My thanks to all those who
participated in the hours of discussion and analysis of the
issues of biochemistry as well as issues of a broader scope.

The encouragement and faith of my parents and my
wife's parents is appreciated.

Finally. my most sincere thanks and appreciation to
my wife, Shirley, who typed so very many theses and drew so
very many figures to make this final thesis possible. With—
out her love. encouragement and spirit this thesis and
research could not have been done. I thank my son. Troy.
for his endless patience and for his help in caring for his
sister, Reyna.

The financial assistance of the National Science

Foundation and the Department of Biochemistry. Michigan
State University is appreciated.

iii

rp———'

TABLE OF CONTENTS

Page

INTRO wCTION I I I I I I I I I I I I I I I I I I I I 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . 9

The cCg-Photosynthetic Carbon Reduction

I I I I 1 o
The C -Dicarboxylic Acid :Pathway of Photo-
s thesis . . . . . . . . . . . . 13

Location of C-4 Pathway . . . . . . . . . 15

leaf Anatomy and Chloroplast Structure . . . 18

Photosynthesis Rates . . . . 19

C02 Compensation.Point and Oxygen Inhibi-
tion of Photosynthesis . . . . . . . . . . 20

Environmental Correlations . . . . . . . . . 20

Glycclate Pathway . . . . . 22
3-P-Glycerate Metabolism and. Glycerate

Fomtlon I I I I I I I I I I I I I I I I 2?

Glycerate Formation . . . . . . . . . . 29

Acid Phosphatases from Leaves . . . . . . . . 32

nTERan AND mfioDs I I I I I I I I I I I I I I I 38

P nuts I I I I I I I I I 38

Chemicals . . .

Protein and Chlorophyll .
Phosphate Determinations
Glycerate Determinations
Enzyme Assays . . . . .
Substrates . . . . . .
Isoelectric Focusing .
Gel Filtration . . . . .
Ion Exchange Chromatography
Paper Chromatography . . .
Preparatory Methods . . . .

“Mme I I I I I I I I I I I I I I I I I I I I
PART A: 3-P-GLJCERLTB PHOSPHLTASE PROM SUGARCANE
LENVES

I I I I I I I I I I I I I I I I I I 51

 

I I I I I I I I I I I I
I I I I I I I I I I I I
I I I I I I I I I I I I
I I I I I I I I I I I I
I I I I I I I I I I I e
I I I I I I I I I I I .
I I I I I I I I I I I I
I I I I I I I I I I I a
I I I I I I I I I I I I
6;?

   
  

I
I

U!
ps

I. Purification of Sugarcane 3-PuGlycerate
P haunt... I I I I I I I I I I I I I I I I I 51

Extraction from Sugarcane Leaves . . . . . . 51
pH PrIItlonItlon e o e e e a e e e e e e e e 53
iv

II.

III.

 

  
 

PART B:

I.

First Acetone Fractionation . . .
Ammonium Sulfate Fractionation .
Second Acetone Fractionation . .
Sephadex G-200 Gel Filtration . .
Chromatography on Phosphocellulose .
Chromatography on Carboxymethyl Sephadex
Concentration of Enzyme. . .
Discussion of the Purification Procedure

Biochemical Properties of 3-P-Glyoerate

The Enzyme Assay . . . .
pH Optimum . . .
Kinetic Characteristics .
Effect of Temperature . .
Isoelectric Point . . . .
Effect of Ionic Strength .
Substrate Specificity . . . .
Effect of Metal Complexing Agents .

h

Effect of Cations . . . . .
Effect of Related Compounds and 0t e
Inhlbl t Ors I I I I I I I
Inhibition of Glycidol-P . .
Effect of Phosphonic Acid Derivatives
Products of the Enzyme Reaction . . .

ofieeeeeeeee

e e 0 e
e e e e
e e a e

Physical Characteristics . . . . . . .

Sucrose Density Gradient Centrifugation
BBS-Polyacrylamide Electrophoresis . . . . .
Polyacrylamide Gel Electrophoresis . . . . .

Physiological Considerations of 3-Phospho-
glycerate Phosphatase in Sugarcane Leaves . .

Location in the Sugarcane Plant . . .
Localization of the Sugarcane 3-P-Glycerate
Phosphatase in leaf Tissue . . . .
Localization of 3-PuGlycerate Phosphatase
by NonnAquecus Density Fractionation . . .
Development of the 3-PdGlycerate Phosphatase
in Etiolated Sugarcane After Illumination.
Diurnal Variation of the Enzyme Activity . .

THE DISTRIBUTION OF 3-P-GLICERATE AND
P-GLICOLATE PHOSPHATASE IN VARIOUS PLANTS .

Special Materials and Methods . . . . . . . .
Results and Discussion . . . . . . . . . . .
Relative Levels of 3-P-Glycerate and
P-Glycolate Phosphatases as a Function of

the Rate of Photosynthesis in Soybeans . . .

V

11?

120
120
121
126
127
131
136
137
138

M9

 

 

PART C:

FROM SPINACH LEAVES .

I

A PARTICULATE 3-P-GLYCERATE PHOSPHATASE

PuGlyoolate Phosphatase in Chloroplasts . .
BdeGlycerate Phosphatase in Starch Pellet

pH Optimum

Kinetics of Enzyme
Effect of Divalent Cations
Substrate Specificity . .

I

Solubilizing the Particulate BlP-Glycerate

Phosphatase

Purification of the Starch Particle
Glycerate Phosphatase

DISCUSSION . . .
SUMMARY . . . .
BIBLIOGRAPHY . .
APPENDIX A . . .
APPENDIX B . . .

 

V11

3-P-

177
201
205
215
216

 

Figure

 

LIST OF FIGURES

Schematic Representation of the C -Dicar-
boxylic Acid Pathway of Photosynthetic C02
‘ssimi lat ion I I I I I I I I I I I I I I I I

G 1y°°1£t° P athway e e c s o e e e o e o e a

Schematic Relationship Between Chloroplasts,
Peroxisomes and Mitochondria with Regard
to Glycolate Metabolism . . . . . . . . . .

The Elution Pattern for Sephadex G-200 Gel
F 1 1trat ion I I I I I I I I I I I I I I I I I

The Eluticn Pattern of the Phosphccellu-
lose Ion Exchange Column . . . . . . . . . .

The Elution Pattern of the Carboxymethyl-
Sephadex Ion Exchange Chromatography . . . .

The Percentage of the Enzymes Solubilized
as a Function of Homogenization Time or
U80 Of the Roller M111 I I I I I I I I I I I

The Stability of 3un61ycerate Phosphatase
as Function of pH and Ionic Strength . . . .

3-P-Glycerate Hydrolysis by the PhOSphatase
as a Function of Time and EnZyme Concentra-

tion I C . C C I I I O I I I I I O I I I I C

The pH Activity Curves for 3-P-Glycerate
Pholphatase.o..............

The pH.Activity Curve for 3~P-Glycerate
Phosphatase and PuGlycolate Phosphatase
Using First Acetone Fractions . . . . . . .

The Relationship of Substrate Concentration
to Reaction Velocity and a Lineweaver-Burk
Plot for 3-P-Glycerate Phosphatase . . . . .

Effect of Temperature of 3-PaGlycerate
PhOBphItIBI Stability and Rate of Hydroly-
8180f3-P-Glycorat9............

vii

Page

25

56

59

61

65

73

76

78

81

an

T"'_—————

Figure Page

14 The Isoelectric Focusing Profile of 3-P-
Glycerate Phosphatase . . . . . . . . . . . . 87

15 The Effect of Ionic Strength on 3-P-
Glycerate Phosphatase Activity . . . . . . . 90

16 Lineneaver-Burk Plot of L(+)Tartrate Inhi-
bition I I I I I I I I I I I I I I I I I I l 1 on

17 Time Dependence of En2yme Inactivation by
GmidOI-Peeeeeeeeeeeeeeeee106

18 Lineueaver-Burk Plots of Glycidol-P Inhi-
bition I I I I I I I I I I I I I I I I I I I 107

19 The Michaelis Curve for 3-P-Glycerate Phos-
phatase in the Presence of Phosphonic Acid
Derivatives . . . . . . . . . . . . . . . . 110

20 Sucrose Density Gradient Profiles . . . . . . 113

21 Gel Scan of SUB-Polyacrylamide Gel Electro-
phoresis of 3-P-G1ycerate Phosphatase . . . . 118

22 Molecular Weight of Polypeptide Chains of
3spsslycerate Phosphatase . . . . . . . . . . 119

23 3-P-Glycerate Phosphatase Deve10pment from
Etiolated Sugarcane Tissue Upon Illumination. 130

2“ Diurnal Variation of 3-P-Glycerate Phospha-
tase Activity . . . . . . . . . . . . . . . . 132

25 Extraction Time Versus Percent of the Phos-
phatase Activity Sclubilized for 3 Types of
Plants I I I I I I I I I I I I I I I I I I I 1&5

 
 
 
 
 
 
 
 
 
  

 

26 The Activities of 3-P-Glycerate and P-Gly—
colate Phosphatases Versus Rate of Photo-
synthesis in Soybean Varieties . . . . . . . 151

27 The pH Activity Curve for 3-P-Glycerate
Phosphatase in Starch Particles . . . . . . . 161

28 Linearity of the 3-P-G1ycerate Phosphatase
‘ss" I I I I I I I I I I I I I I I I I I I I 1 61

29 Kinetic Plots for the Spinach Particulate
BqP-Glycerate Phosphatase . . . . . . . . . . 163

30 Solubilization cf the Starch Grain 3-P-
Glycerate Phosphatase as a Function of pH . . 169

viii

pd‘ I

  

Solubilization of the Starch Grain 3-P-

Page

Glycerate Phosphatase by B-Anylase . . . . . 169

Solubilization of the Particulate 3-P-
Glycerate Phosphatase as a Function of
Salt Concentration .

Proposed Intercellular Movement of Newly
Fixed Carbon Between Bundle Sheath and
Mesophyll Cells

 

. 172

. 192

 

 

II

III

VI

VII

VIII

XI

 

 

Table

LIST OF TABIES

Acid Pathway Between Two Typ s of Chloro-

Looation of Enzymes of the 03-Dicarboxylic
ph8t8(16.u1)eeeeeeeeeeeeeo

Summary of the Purification of 3-P-Glycer-
ate Phosphatase from 840 g Sugarcane Leaves

Specificity of 3-P-Glycerate Phosphatase .

Relative Specificity of 3-P-G1ycerate Phos-
phatase at Various Stages of Purification .

Activity of 3-P-Glycerate Phosphatase with
Two Substrates Present . . . . . . . . . .

The Effect of Metal Complexing Agents on
3-P-G1ycerate Phosphatase Activity . . . .

Effect of Metal Ions on 3-P-Glycerate Phos—
thase I I I I I I I I I I I I I I I I I I

Effects of other Inhibitors and Related
C amp ound s I I I I I I I I I I I I I I I I I

Hydrolysis of Phosphonic Acid Derivatives

and Their Effect on 3-P-Glycerate Hydrolysis

by 3-P-G1ycIrItI PhOSphatase e e e e e e e
Stoichicmetry of 3-P-Glyceric Acid Phospha-

88' I I I I I I I I I I I I I I I I I I I

Distribution of 3-PuGlycerate Phosphatase in

Sugaroam P but I I I I I I I I I I I I I I

Distribution of 3-PaGlycerate and P-Glyco-
late Phosphatase in Isolated Sugarcane
Chloropla8t8.........o....o

Fractional Centrifugation of an Isotonic
Extraction of Sugarcane Leaf Tissue . . . .

Distribution of 3-P-Glycerate Phosphatase
After Fractional Centrifugation for Isola-
tion of Percxiscmes . . . . . . . . . . . .

I

Page

17

52
91

93

95

97

98

101

109

111

120

123

125

126

r.___—_

Table Page

XV Distribution of Chlorophyll and Encymes
after NonpAqueous Isolation and Density
Fractionation of Destarched Corn Leaves . . . 128

XVI The Levels of Activity and the Distribu-
tion of 3-P—Glycerate Phosphatase and
P-Glycolate Phosphatase in Various Higher
Plants, Algae and Liverwort . . . . . . . . . 139

XVII Correlation of 3-P-Glycerate Phosphatase
and P-Glycolate Phosphatase Activities with
Photosynthesis in 15 Soybean Varities . . . . 153

XVIII Phosphatase Activity of Isolated Spinach
Chloroplasts Prepared in Sorbitol Medium
as Described in Materials and Methods . . . . 15?

XIX Distribution of 3-P-Glycerate Phosphatase
in Peroxiscme Preparation . . . . . . . . . . 159

XX The Effect of Divalent Cations on the
P BAP-Glycerate Phosphatase in Starch Particles 165

XXI Substrate Specificity of the Starch Particle
3-P-Glycerate phosphatase . . . . . . . . . . 167

XXII Purification of the Particulate 3-P-Glycer-
ate Phosphatase from 300 g of Spinach Leaves. 175

 

xi

 

 

  

Ax

c-KG

AMP . ADP . ATP
Chl

CM-

DEAE-

DHAP

BETA

f.c.

Hepes

PEP
P-Glycolate

BAP-61 cerate
(3sPGA)
VP. P1. PPi

LIST OF ABBREVIATIONS

Absorbance at wavelength 1
a-Ketoglutarate

Adenosine mono-. di-, and triphosphate
Chlorophyll

Carboxymethyl-

Diethylaminoethyl-

DihydroxyacetOne phOSphate

Ethylenediaminetetraacetic acid
(disodium salt)

Foot candles

N—Z-hydroxyethylpiperazine-N'-2-ethane
sulfonic acid

Michaelis constant
2—(N-morpholine)ethanesulfonic acid

Nicotinamide-adenine dinucleotide
(reduced)

Nicotinamide-adenine dinucleotide
phosphate (reduced)

Oxaloacetate
Phosphoenol pyruvate
Phosphoglycolate
34Phosphoglycerate
PhOSphorous, inorganic phosphate.
pyrophOSphate
Ribulose-1.5-diphosphate

xii

 

Specific activity

Trichlorcacetic acid 1;]

” Sodium dodecyl sulfate ‘ 7 if"
‘1“
ms-

.Triethylamino ethyl— . 1
his N-tris( hydroxymethylhethylglyc ine . f

        

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If someone tells me tmt in making these conclusions
I have gone beyond the facts I reply: 'This is true. that
I have freely put myself among ideas which cannot be rigor-
ously proved. That is my way of looking at things. Every
time a chemist concerns himself with these mysterious
phenomena and every time he has the luck to make an impor-
tant step forward he will be led instinctively to attribute
their prime cause to a class of reactions in harmony with
the general results of his own researches. That is the
logical course of the human mind, in all controversial
matters.’

.. Louis Pasteur, 1857

xiv

 

 

 
 

INTRODUCTION

The last quarter century of research in photosynthe-
sis since Calvin's group envisioned and proposed the "path
of carbon in photosynthesis" (1, 2, 3) has seen the elucida-
tion of many of the enzymes. intermediates and products of
the pathway (1+, 5. 6. 7). The process has subsequently been
called "the photosynthetic carbon cycle," or Calvin's cycle.
or more recently the c-3 pathway. The kinetic isotope tracer
studies allowed the determination of the sequential formation
of intermediates and products. Enzymological studies have
revealed substrates (NADPH and ATP) which were unlabeled in
radictracer eXperiments and provided information regarding
regulation of the pathway (8, 9. 10. 11. 12). The initial
product of the Calvin cycle is the 03 acid, 3-P-g1yceric acid
(13). The subsequent metabolism of 3-P-glycerate regenerates
the cog acceptor. ribulose—i,5-diphosphate. through a
sequence of reactions involving the sugar mono- and diphos-
phates. This pathway of photosynthetic 002 fixation was
considered to be ubiquitous in higher plants and algae.

However the 63-pathway has failed to explain all the
products formed during 11"002 incorporation by many photosyn-
thetic organisms. The novel pathway of 602 fixation which
involves the ferridoxin dependent reductive carboxylation cf
eestyl-CoA and succinyl-CoA to yield pyruvate and a-keto-
1

r,.__—

2
glutarate is termed the reductive carboxylic acid cycle and
appears to be limited to two heterotrophic photosynthetic
bacteria (14). The very early and rapid incorporation of
1”c02 into the dicarboxylic acids, malate and aspartate, of
sugarcane and corn was initially ignored. but the persistence
of this occurrence stimulated a thorough investigation of the
photosynthesis of sugarcane and corn (15. 16. 1?. 18). The
subsequent radiotracer experiments and enzyme studies have
confirmed that a second unique process of 002 assimilation
} termed the Cu-dicarboxylic acid pathway (Cu-pathway) is
cperative in the tropical and sub-tropical grasses such as
} sugarcane as well as in other species (Figure 1) (16).

The C3-pathway of C02 assimilation also fails to
explain the early and rapid incorporation of 1“,002 into
glycolate (d-hydrcxy acetate) (5. 19. 20). The amount of
newly fixed carbon dioxide found in glycolate and the
"glycolate pathway" (Figure 2) ranges from 0 up to 92% of
the total carbon fixed (21, 22), and is dependent on several
physiological and photosynthetic conditions to be cited in

 

the Literature Review. The accumulation of glycolate and

     
   
 
 
 

its products is observed especially with young or rapidly
growing tissue. Thus it seems that glycolate should have a
very significant role in photosynthesis.

V Glycolic acid is believed to be biosynthesized from
some intermediate(s) of the C3-pathway and its metabolism is
closely related to photosynthetic conditions (22). P-Glyco—
late is believed to be the immediate precursor in the

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6
biosynthesis of glycolate, since it is hydrolyzed by a
specific P-glycclate phosphatase in the chlorOplasts (23, 2h).
Recent investigations in this laboratory on glycolate metabo-
lism have established that some of the glycolate metabolizing
enzymes are compartmentalized in microbodies termed leaf
percxisomes (25. 26). Available evidence strongly supports
the view that the leaf peroxisomes and the glycolate metabo-

   
 
 
    
   
   
  
   
  
  
 
  

lism therein are responsible for the Cog—photorespiration
(peroxisomal reapiration) observed with many higher plants.
A survey of both COz-photOFOSplrlng and noanOZ-photcrespir-
ing plants was made by Tolbert's group (26) for peroxisomes
and glycolate metabolizing enzymes. P-Glycclate phosphatase
was included in the survey since the phosphatase is presumed
to be necessary for glycolate biosynthesis. When surveying
for P-glycolate phosphatase. 3-P-glycerate was used as an
indicator of substrate specificity and relative phosphatase
levels. Sugarcane, a nonpcoz-photorespiring plant. possess-
ing the 04-dicarboxylic acid pathway of 002 fixation. had
low levels of P-glycolate phosphatase paralleling the low
levels of the other glycolate metabolizing enzymes and

peroxiscmes. However. extracts of the sugarcane leaves

 

hydrolyzed 3-P-glycerate much more rapidly than P-glycolate.
Hydrolysis of 3-P-glycerate in crude extracts of other

plants (cog-photorespiring plants) was usually at a rate 25
to 30% of that found for P-glycolate. Since glycerate is a
major product of glycolate metabolism (27) and the glycolate
metabolizing enzymes were low in sugarcane. we felt that the

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7
rapid enzymatic hydrolysis of 3-P-g1ycerate might be indica-
tive of a bypass of the glycolate pathway. A 3-P-g1ycerate
phosphatase would also eXplain the lacoz incorporation into
the carboxyl position of glycerate during photosynthesis
giving an isotOpic distribution inconsistent with the glyco-
late pathway (28, ‘29). The carboxyl-labeled glycerate

found during photosynthesis experiments had previously been
attributed to inadequate killing procedures of the experi-
ment and was believed to be insignificant, even though in

some leaves large metabolically active reservoirs of glycerate
were present (7, 28, 29).

The following points suggested that an investigation
of a BAP-glycerate phosphatase would contribute significantly
to the understanding of the path of carbon in photosynthesis
and photorespiration. (a) Glycolate metabolism and P-glyco-
late phosphatase activities were low in sugarcane. (b) Enzy—
matic hydrolysis of 3-P-g1ycerate by sugarcane extracts was
unusually rapid. (c) Sugarcane is a non-Coz-photorespiring
plant with a C02 assimilation process differing from the
normal Calvin cycle from which glycolate is made. (d) A
BJP-glycerate phosphatase would provide a bypass of the gly-
colate pathway (negating the need for it). thus bypassing
the source of respired 002 and 02 uptake of Cog-photorespira-
tion and explaining the low level of glycolate metabolism.
(e) A 3-P-g1ycerate phosphatase would explain the appearance
of carboxyl-labeled glycerate (and serine) in 14002 photo-

'synthesis experiments.

 

 

8

The purification and characterization of 3-P-glycer—
ate was undertaken to determine if this enZyme was similar
to P-glycolate phosphatase with reapect to such parameters
as specificity. location and cofactor requirements. Such
similarities would support our conclusion that 3-P-glycerate
phosphatase was a substitute for P-glycolate phosphatase and
in addition provide possible information useful in under—
standing the function of both phosphatases. Studies directed
towards the formation. location. and physiological changes
in activity would be useful in delineating whether or not
the 3-P—g1ycerate phosphatase was involved in photosynthetic
processes. Studies comparing the distribution of both
phosphatases in the plant kingdom should help to elucidate
their true function(s).

This thesis describes the study of 3-P-g1ycerate
phosphatase of sugarcane leaves. Procedures for the partial
purification of the enzyme and some of its biochemical and
physical properties are described. The distribution of
this phosphatase. as well as that of P-glycolate phosphatase
among many plants, is also presented as well as some physio-
logical studies of its presence in leaves. A study attempt-
ing to correlate the relative levels of 3-P-glycerate phos-
phatase and P-glycolate phosphatase to photosynthetic rates
in soybeans is presented. The discovery and partial charac-
terization of a particulate (probably starch grains) 3-P-

glycerate phosphatase from spinach leaves is also presented.

 

  

LITERATURE REVIEW

The studies on 3-P-glycerate phOSphatase are directed
ultimately towards determining its function as a part of the
photosynthetic process of green plants. Since the source of
this enzyme is the photosynthetic tissue of sugarcane. and
its substrate is the first or one of the first major inter-
mediates of photosynthetic C02 assimilation. the two major
pathways of photosynthetic carbon metabolism will be
reviewed. The functions to be attributed to the 3-P-glycer-
ate phosphatase require a review of the glycolate pathway
and photorespiration. Consideration of the importance of
the 3-P-glycerate to the carbon metabolism of the leaf
requires a review of 3-P-g1ycerate metabolism and glycerate
formation. Obviously, Justice cannot be done to each of
these exciting and dynamic research areas without writing a
very large book: however, the author feels a brief review
of these areas will facilitate the discussion of the studies
on BAP-glycerate phosphatase in leaves.

The reductive assimilation of free atmoSpheric carbon
by photosynthetic organisms through utilization of light

energy is summarized by the equation:

hv
nco2 + anszo ———> n02 + (0320):: + nHZO

.....s.

.I. I
...

 

   

10
and is termed photosynthesis. This complex process entials
converting light energy to chemical energy of excited chloro—
phyll molecules. converting this chemical energy to reducing
equivalents. NADPH and high energy pthphate, ATP, and reduc-
ing 002 to carbohydrate. using the reducing power and ATP.
The "path of carbon in photosynthesis" describes how the ATP
and NAIPH are used for 002 assimilation in green photosyn-
thetic organisms (30).

The C3-Photosynthetic Carbon Reduction Cycle

 

Radicautography of chromatographed extracts from the
green algae, Chlorella and Scenedesmus, which had been

 

supplied with luccz for less than 5 sec during steady state
photosynthesis, revealed carboxyl-labeled 3-P-glycerate as
the first labeled compound of photosynthesis (13). Since
002 is the only source of carbon, the acceptor molecule

must be regenerated, thus a cyclic pathway is inherent.
Through an elegant series of experiments (13. 31. 32. 34)
involving light and dark transitions coupled with a knowledge
of metabolic pathways of glycolysis and pentose phosphates,
Calvin's group established that the carbon reduction cycle
involves (a) the carboxylaticn of ribulose 1.5-diphosphate
to give two molecules of 3-P-glycerate [or 3-P-glycerate

and triose phosphate (3h)], (b) the reduction of 3-P-glycer-
ate to triose phosphate. (c) the condensation and conversion
of triose phosphates to hexose phosphates and pentose phos-
phates, and (d) the regeneration of ribulose diphosphate

 

  

11
from pentose phosphates and ATP (30). The adequacy of the
03-pathway as a means of 002 fixation was presented by
Bassham and Kirk (35) who found that upwards of 85% of the
C02 fixed by Chlorella could be accounted for by the C3-
pathway and its immediate products. Massive evidence for
the C3-pathway has been accumulating since the pathway was
proposed in the early 1950's (3“, 38). The isolation of
intact spinach chlorOplasts capable of C02 fixation and
carbon cycling at close to 12.3112 rates has been instru-
mental in localizing the C3-pathway in the chloroplasts
(36. 37. 38).

The enzymes for the C3-pathway have been found sup-
porting the radiotracer experiments (10. 11. 30. 38). The
nonaqueous isolation and density gradient centrifugation
studies by Stocking (39), Smillie (40) and others (#1) have
provided excellent evidence for the localization of the C3-
pathway enZymes and intermediates in the chloroplasts. Con-
trol of the 03-pathway is manifested through light dependent
activation of enzymes, formation of metabolites or changes
in the energy charge. Control is also manifested by changes
in substrate or cofactor levels (feedback inhibition and
hcmotrcpic interactions) (12).

The function of the C3-cycle is to provide carbon
compounds to the plant. Although the chloroplast is capable
of making all the major types of compounds. there are some
priorities necessary in the photosynthetic products in order
for the plant to be able to survive. Except for the formation

m

New.

I‘i.

.Nfil

”I‘ve

 

 
 

12
of glycolic acid, some amino acids and certain carbohydrates
which will be discussed later in the review, sucrose and
starch (a-glucan) are the major immediate products in the
photosynthetate. Sucrose and starch represent a sink or
reservoir for carbohydrate produced from the energy and
reducing power provided by the photochemical event. Both
are made in the chloroplast (#2, 43, an). Sucrose is the
chief carbohydrate transported elsewhere in the plant (nu).
Chloroplast starch or assimilatory starch is used during
low light periods or darkness for biosynthesis and energy.
Starch synthesis is regulated by intermediates of the 03-
pathway, 3AP-glycerate and fructose-6-phoSphate, at the AIP-
glucose pyrophosphorylase level (12).

The C3-pathway has been found in numerous higher
plants, green algae and photosynthetic bacteria. The C3-
pathway was assumed to be ubiquitous, until evidence for the
reductive carboxylic acid cycle in some photosynthetic
bacteria was recently reported (14), and a second major
pathway (Cu-pathway discussed below) was proposed for higher
plants (16). The belief that the C3-pathway was the univer-
sal pathway was partially due to geography and choice of
plant material. Most research was done in the temperate
geographic zones, using easily manipulated tissue such as
spinach, soybeans, and green algae. These plants give
perfect examples of the C3-pathway. It is almost incredible
as one reads the literature of photosynthesis with the
present information that 99% of the earlier photosynthesis

13
research was done using a limited number of temperate zone
plants. Corn and sugarcane, which are extremely important
economic crops, and should have been studied, were too dif-
ficult to use in photosynthesis experiments because of their
tough leaf tissue and consequently were assumed to possess
the C3-pathway of 002 assimilation. Sugarcane and corn
have an exceptional capacity for producing dry matter (#5)
and are capable of photosynthesis rates much greater than
most other plants (46, #7).

The Cu-Dicarboxyl Acid Pathway of Photosynthesis

 

Tarchevskii and Karpilow (50) in 1963 and Kortshak.
Hartt and Burr (15) in 1965 noted that aspartate and malate
were the first compounds labeled during 14002 experiments
with corn and sugarcane.* Hatch and Slack extended these
experiments and added oxaloacetate to the list of initially
labeled compounds (17). Using sugarcane, corn and sorghum
leaves, radiotracer photosynthesis studies by Hatch and
Slack showed that:

(a) Incorporation of 1“C into oxaloacetate, aSpartate

and malate was linear with time, light dependent

luCOZ-fixation (17, 18,

and occurred first during
51).

(b) The 1“c labeling into c-u of dicarbcxylic acids
was faster than the C-1 labeling of 3-P-glycerate

(51).

 

 

    

*Calvin's group back in the 50's had similar data,
_ but ignored it! (Personal communication from one who was
. _ present.)

\A’

3.!

in

(c) The 14C in C—4 of the dicarboxylic acids became
the C-1 of 3-P-glycerate in a light dependent
reaction, and then flowed sequentially through
dihydrcxyacetone-P, the hexose phosphates, and
the pentose phosphates to sucrose and d-glucan
in a manner consistent with their role as inter-
mediates in the fixation cycle (51).

1“'0 accumulated in Hull3 when leaves fixing 11+C02

(d)
were placed in COZ-free air (51), thus suggest-
ing it was the 002 acceptor from the C-# of the
dicarboxylic acid or an immediate precursor.

With the above evidence Hatch and Slack proposed the
cyclic Cu-dicarboxylic acid pathway in Figure 1. The mode
of transferring the C-h of the dicarboxylic acids to C-1 of
3-P-g1ycerate is uncertain at this time. The enZymes for
all the reactions except the "transcarboxylase" reaction
have been established as being present in adequate amounts
(1+1. 52. 53. 51+. 55).

The enzymes found include three enZymes unique to the
04-pathway of photosynthesis, phosphoenol pyruvate (PEP)
carboxylase for the primary carboxylaticn reaction (52);
pyruvate, P1 dikinase to regenerate the primary C02 acceptor,
PEP (5#, 56); and NMIP-malate dehydrogenase to reduce oxalo-
acetate (41).

Attempts to isolate an enzyme catalyzing the proposed

 

  

transcarboxylation have been unsuccessful (16. 57). Andrews
(5h) proposes that the transcarboxylation process is the

 

 
 

15
coordinated result of malic enzyme (52, 55) and RuIP carboxyl-
ase. The oxaloacetate made by the PEP carboxylase would be
converted to malate by the NADP—malate dehydrogenase. The
malate would be decarboxylated in the presence of RuDP car-
boxylase by malic enZyme (NAIF-malate dehydrogenase decar-
boxylase). The C02 (possibly enzyme bound) would be
refixed by the RuDP carboxylase to yield 3-P-glycerate. The
RuDP carboxylase activity, which was initially reported as
being too low to have an important role in the Cu-pathway,
has been found to be adequate and to possess even better
kinetic parameters than the enzyme in C3-pathway plants
(5“. 58).

Location of C—# Pathway
Corn, sugarcane and other Cu-pathway plants differ

from C3-pathway plants in that they contain two morpholog-
ically distinct types of chloroplasts. Roades and Carvalho
(#8) and Brown (119) noted that panicoid grasses had two
different types of chloroplast containing cells. These
chloroplasts were of two distinct types with differing
‘starch storing capacities (#8, #9), but the significance

of this to the photosynthetic process in these plants was
not considered until recently when the Cu-pathway was
thoroughly investigated. The two types of chloroplasts
have been designated mesophyll chloroplasts and bundle
(parenchyma) sheath chloroplasts by their presence in these
cells. The bundle sheath chloroplasts contain the assimila-
~tcry starch. Using nonpaqueous isolation and density

 

 

16
fractionation techniques, Slack and associates (#1, 59) were
able to separate bundle sheath chloroplasts and mesophyll
chloroplasts by their differences in starch content. This
permitted him to distinguish en2ymes of mesophyll chloro-
plasts from bundle sheath chloroplasts and also chlorOplas-
tic enzymes from cytosol. This same procedure could be
used to localize labeled intermediates produced in 1“C02
experiments. The results of these experiments are summar-
ized in Table I.

All the enzymes of the regular C3-pathway of photosyn-
thesis plus malic enzyme are located in the bundle sheath
chloroplasts (Table I), while the enzymes of the Cu-pathway
are localized in the mesophyll cells. According to Andrews
(5#), the fact that malic enzyme (high activity only in C#'
pathway plants) was located in the bundle sheath chloroplasts
supports the proposal that it is part of the transcarboxyla-
tion mechanism. It would also provide half of the reducing
power needed to convert 3-P-glycerate to triose phoSphates,
since there appears to be a low level of reducing power in
the bundle sheath cells (57).

The 11*c labeled intermediates of the Cu-pathway were
located in the plastids with their respective enzymes.
3éP-Glycerate and DRAP were distributed fairly evenly between
the two types of chlorOplasts (#1).

In sumary the investigators in Australia have pro-
vided the following evidence for the Cu-pathway of photo-
synthesis in a large number of plants (57, 60).

 

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['1‘

u.

 

  

18
(a) The labeling of the primary carboxylaticn products
were light dependent and showed the necessary
kinetics of labeling.
(b) The 1["0 flowed sequentially through the proposed
cycle.
(o) The labeled intermediates were found in the
chloroplasts.
(d) The enzymes necessary were present in adequate
amounts and were in the plastids.
(e) The enzymes were synthesized during the biogenesis
of the chloroplasts in corn plants.
(f) The pathway operates in a cyclic manner to regen-
erate the CO2 acceptor moiety.
It has been mentioned above that there are certain
morphological, physiological, and environmental correlations

between which plants have the C3- or Cu-pathway.

leaf Anatogz and Chloroplast Structure
Three sub-families of Gramineae, Panicoideae,

Aristoideae and Chloridoideae, have their leaf vascular
bundles sheathed by large, chloroplast-containing, parenchyma
sheath cells which are surrounded in a radial fashion by meso-
phyll cells (with small and numerous chloroplasts) (#8, #9).
The chloroplasts of the bundle sheath cells accumulate con-
siderable starch. Dicctyledons such as the Amaranthus of

the Amaranthaceae and some Atriplex of the Chenopodiaceae
possess the same leaf anatomy (61). Without exception,

 

   

 

19
plants with these features that have been investigated have
the Cu-pathway. For intragenera exceptions such as Panicum
of the Panicoideae in which the Cu-pathway was missing and
only the Cg-pathway was present, the bundle sheath cells
lack developed chloroplasts (57).

The chloroplasts of the mesophyll cells have the
familiar grana stacks. Chloroplasts of the bundle sheath
cells in the Panicoids are without grana but grana are
present in the bundle sheath chloroplasts of some other
Cu-pathway plants. The two types of cells of the Ch-pathway
plants are connected by numerous plasmadesmata (62). A well
developed peripheral reticulum linked directly to the bound-
ing membrane of the chloroplasts was also noted by Laetsch
(62) and others (63) and it has been suggested that this
peripherial reticulum may interconnect the two types of
chloroplasts (57, 62).

Photosynthesis Rates
Photosynthesis rates of Cu-pathway plants are high,

averaging 60-70 mg 002 per dm2 per hour as opposed to 5-30
mg 002 per dm? per hour for C3-pathway plants. The photo-
synthesis of 03-pathway plants in air is saturated at less
than 50% of full sunlight while photosynthesis of the C#'
pathway plants remains unsaturated even at full sunlight
(#6).

.f‘.

 

20
002 Compensation Point and Oxygen

Inhibition of Photosynthesis
The 002 compensation point is the 002 concentration

 

at which the rate of photosynthesis equals the rate of car-
bon dioxide released by respiration. Cu-pathway plants

show a C02 compensation point of less than 5 ppm 002 (6M) and
can reduce the 602 concentration of a closed system nearly
to zero (65). C3-Pathway plants show a finite value in the
15-150 ppm 002 range, however at low 02 pressures their com-
pensation point approaches that of 04-pathway plants in air
(65).

Photosynthetic coz fixation in plants with the C3-
pathway is inhibited by 02 at its concentration in air (21%)
(67). This inhibition decreases with decreasing oxygen con-
centrations (67) and has been attributed to Cog-photoreSpira-
tion which will be discussed below with glycolate metabolism.
In the 04-pathway plants there is no effect of oxygen on
rates of net photosynthetic C02 fixation (65).

Environmental Correlations

Plants with the Cu—pathway inhabit high sunlight areas
of the world, such as grasslands, open forests and arid or
semi-arid regions (#9. 57). The inter— and intragenera
exceptions to the Cu-pathway distribution support this corre-
lation. The Panicum of the Panicoideae inhabits the shaded
rain-forest floor and is a C3-pathway plant instead of a Cu-
plant as are most other panicoids (57). Bamboo occurs in

high sunlight and tropical regions but is a C3-plant (57).

 

 
 

21
However, the habitat of bamboo receives adequate rainfall
distribution over the whole year. CuéPathway plants would
definitely have an advantage in areas of frequent or annual
drought. Their more efficient net 002 fixation (no loss of
002 from photorespiration) permits much shorter periods of
open stomata from which vital water would tranSpire.

The examples of the above correlations are plentiful
and such data are increasing. There is no taxinomic evi-
dence for the Cu-pathway in primitive plants or algae. The
Cn-pathway plants are located among the most highly evolved
plant species.

The fundamental differences between the Cu-pathway
and tin C3-pathway are the reactions involving the incorpor-
ation of C02 into 3-P-g1ycerate, the location of the carboxy-
lation reactions, the correlation between the C3-pathway and
C02 photorespiration, the lack of the C02 photorespiration
in Cu-plants, and possibly the stoichiometry of the pathways
with regard to ATP and NAIPH2 requirements which has yet to
be resolved (16, 57). The major, quantitative and products
of both pathways are sucrose and assimilatory starch.

In both the 03— and Cu-pathway, 3-P-g1ycerate is a
major early product of photosynthesis which is then photo—
synthetically reduced to sugars. What we believe to be the
reasons for its hydrolysis by a specific phoSphatase have
already been presented. First it seems necessary to under-
stand the properties of this phosphatase and some of its
physiological characteristics. This is the objective of
this thesis.

I.
n-

22

Glypolate Pathway

Any consideration of compounds resulting from photo-
synthesis certainly must include the glycolate pathway. The
glycolate pathway describes the metabolic sequence from P-
glycolate and glycolate through glyoxylate, glycine, serine
and hydroxypyruvate to glycerate (22, 27). Glycolate and
the intermediates of the pathway (Figure 2) are rapidly

-.Av .._. a- u—a. _‘ ...—N‘—

labeled during lucoz photosynthesis experiments using C3-
pathway plants, however this 1""C incorporation into glyco-
late and products cannot be explained by the C3-carbon
reduction cycle. The mode of glycolate formation during

C02 fixation in photosynthesis remains unknown although 10
to 90$ of the total carbon may be passing through it (21,
22, 69). The kinetics of the 140 accumulation in glycolate
indicate that it is formed from an intermediate of the
C3-pathway (5). The immediate precursor of glycolate is
believed to be P-glycolate formed in the chloroplast (22)
which is hydrolyzed by a specific P-glycolate phosphatase
located in or on the chloroplast (23). Glycolate and the
immediate products formed from short time (#-11 sec) photosyn-
thesis experiments with 11‘002 are uniformly labeled and its
metabolites should subsequently be uniformly labeled. 3-P-
Glycerate and glycerate are carboxyl—labeled at these times.
Feeding experiments with glycolate—1-1% produced glycine-1-
1%, serine-1-1% and glycerate-1-1%. with glycolate-2-1%

 

produced glycine-Z-luc, serine-2,3-1uc and g1ycerate-2,3-1”C
and hBZOlO-1.2.5.6-1uC: with serine-3-140 yielded glycerate-

 

  

23
3-1% and hexose-1.64% (27, 71, 72, 73),

Glycolate formation is definitely linked to photosyn-
thetic conditions. It is favored by low 002 concentration,
high 02 concentrations (20%) and high light intensity (21,
22). Inhibition of photosystem‘ II and manganese deficien-
cies curtail glycolate formation (22, 74). Several of the
enzymes of the glycolate pathway are absent or greatly
reduced in activity in etiolated tissue (75) and develop
during greening (76, 7?). However the manner in which gly-
colate is biosynthesized is a major unresolved part of
photosynthetic carbon metabolism. This in turn contributes
to the fact that the function of glycolate metabolism is
unknown. Although for 20 years glycolate and its metabolism
have been the subject of intense research by several labor-
atories, its role and function in the metabolism of plants
have never been established. The glycolate pathway is
gluconeogenic, ultimately producing sucrose in the light
but yielding malate in the dark (22). Glycolate contributes
to the serine pools of the plant (29) but as will be dis-
cussed later, is not the only source of serine.

Glycolate metabolism appears to be responsible for
photorespiraticn. Photorespiration occurs in the light and
is usually measured as light dependent C02 evolution or
increased 02 uptake. The rate of photorespiraticn has been
estimated at 5 times the dark respiration (78) and is
inhibited by inhibitors of glycolate oxidase (79). The 02
uptake of photorespiraticn is attributed to glycolate

  

 

 
 
 
 

24
oxidation by glycolate oxidase (80), and the C02 released is
attributed to the direct enzymatic conversion of two glycines
to one serine. This gas exchange is the reverse of that
which occurs during photosynthesis. During the last h years
most of the glycolate metabolizing enzymes (Figure 3) have
been found compartmentalized in leaf microbodies, called
peroxisomes (25, 26). Thus glycolate metabolism has been
designated as photorespiraticn or peroxisomal respiration.
The glycolate pathway in peroxisomes and photoreSpiration is
still gluconeogenic with glycerate and C02 as major products.
Glycolate is itself a major product of the C3-pathway of
photosynthesis; it is the substrate for photorespiraticn
(peroxisomal respiration) and it is a major source of glycer-
ate,hexose phosphate and sucrose (27) in C3-pathway plants.
Electron micrographs indicate a close physical association
between chloroplasts, peroxisomes and microbodies (81). It
has been estimated that peroxisomal en2ymes are capable of
as much carbon metabolism as there is C02 fixed during
photosynthesis in the chloroplasts (82). It seems rather a
waste to respire 25$ of the newly fixed carbon, thus reduc-
ing the net photosynthetic 002 fixation and efficiency.
However, no judgment is legitimate until the functions of
glycolate formation and peroxisomal respiration are estab-
lished. A perusal of the glycolate pathway (Figures 2 and 3)
in C3-pathway plants reveals that a 34P-glycerate phosphatase
would be capable of bypassing the glycolate pathway, elimin-
ating the 02 uptake, the 602 evolved and still produce

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27
glycerate. Therefore one might BXpBOt such a phosphatase in
plants lacking the glycolate-peroxisome-photorespiration
phenomenon. Cu—Pathway plants have low levels of glycolate
pathway enzymes, give low yields of peroxisomes, exhibit no
002-photorespiration and have a high net rate of photosyn-
thetic co2 fixation.

QaP-Glycerate Hetabolism.and Glycerate Formation

As discussed earlier, the formation of BAP-glycerate
is ubiquitous during photosynthesis. It is the initial
product of the 03-pathnay, a product of the second, obliga-
tory carboxylaticn in the Cu-pathway, and a dynamic meta-
bolite in both pathways. Presumably the glycolate pathway
will also produce BAP-glycerate during gluconeogenic forma-
tion of hexose and sucrose. This is supported by the dis-
covery of glycerate kinase in the leaf tissue (83) and in
chloroplasts (59). 3-P-G1ycerate has a regulatory role in
photosynthesis as a feedback or product inhibitor of RuDP
carboxylase and as a positive allosteric effector of ADP-
glucose pyrophosphorylase, which in.turn controls the for-
mation of assimilatory starch (12).

During 1“coz photosynthesis experiments with Chlorella

 

the 1"'C: also quickly (10 sec) appears in alanine, malate and
aspartate (8h). The degradation studies of these compounds
show they are heavily carboxyl-labeled (84). The dicarboxylic
acids also have additional label in 0.“ indicating reductive
carboxylations of pyruvate. The BAP-glycerate which is

28
converted to pyruvate through PEP provides substrate for
tricarboxylic acid cycle and acetyl-CoA for lipid synthesis.

Serine is also labeled very early in 1“002 photosyn-
thesis experiments. Serine is rapidly turning over and is
an intermediate in glycolate metabolism. Hess and Tolbert
(29) reported that serine was carboxyl-labe led at short
times (4-11 sec) in tobacco. but less carboxyl-labeled than
3-P-glycerate. By 30 seconds the serine was uniformly
labeled. Since glycine and glycolate (possible serine pre-
cursors) were uniformly labeled they concluded that there
were two routes of serine synthesis. but that serine from
the glycolate pathway predominated. Cinng and Tolbert (28)
using isolated spinach chloroplasts and 1%02 reported the
formation of carboxyl-labeled serine. The reports of
carboxyl-labeled serine suggest tint serine is a product of
either 3-P-glycerate or glycerate from 3-P-glycerate hydroly-
sis.

Cheung 33 _a_l_. (83) found that enzymes for the non-
phosphorylated pathway of serine synthesis (glycerate dehydro-
genase. hydroxypyruvatezL-alanine transaminase) were highest
in green leaf tissue while the enzymes of phosphorylated
pathway (3-P -glycerate dehydrogenase and P-hydrexypyruvate:
L—glutamate transaminase) were highest in seeds. although
there was still enough of the latter in green leaf tissue to
account for a small amount of serine synthesis. Carboxyl-
labeled serine from short periods of 1%02 photosynthesis
can be considered indicative of enzymatic hydrolysis of
3-P-g1ycerate to glycerate. as Babson 23; 31.. suggested (27).

29

glycerate Formation

Glycerate has been.reported many times to be rapidly
labeled in.photosynthesis experiments but its mode of forma-
tion has not been established. Since glycolate is uniformly
labeled in 1""002 photosynthesis. the glycerate that arises
from the glycolate pathway would be uniformly labeled (2?).
when leaves were fed specifically labeled glycolate. glycine
and serine the resulting labeling patterns were consistent
with direct formation of glycerate from serine (27). Glycer-
ate arising from BAP-glycerate during lucoz photosynthesis
should be carboxyl—labeled. Bassham (85) and Heber (86)
have shown that the chloroplast and cytosol pools of BAP-
glycerate are very rapidly equilibrated and the chloroplasts
of C3-p1ants are very permeable to BAP-glycerate in either
direction (87). Data from Slack gt‘gl. (#1) indicates that
the BAP-glycerate in corn.was distributed in both parenohyma
and bundle sheath cells. Thus it is possible to suggest
that enzymatic hydrolysis of BAP-glycerate could occur in
the chloroplasts or the cytoplasm.and still be carboxyl-
labeled at short times in 14002 fixation.experiments. The
appearance of carboxyl-labeled glycerate on radioautographs
of photosynthesis products has often been explained as an
artifact due to enzymatic hydrolysis of BAP-glycerate during
the killing procedure (6). This explanation was supported
by Ullrich (88) who reported that there was considerable
phosphatase action occurring during the killing procedure
with 80% boiling ethanol. Mortimer (89) reported experiments

30
with soybeans which suggested there was considerable in zizg
hydrolysis of BAP-glycerate but that the 80% boiling ethanol
killing procedure was quite adequate. Aronoff (90) reported
carboxyl-labeled glycerate from soybean which was more
carboxyl-labeled than BAP-glycerate after 5 see. This might
be indicative that some of the BAP-glycerate was hydrolyzed
before it had any chance of being metabolized through the
C3-pathway.

Babson_gt a}, (27) found carboxyl-labeled serine and
glycerate with 14002 photosynthesis experiments with leaves
from soybean. coleus. corn. peppermint and barley after 20
seconds in steady state fixation experiments. Corn had the
most carboxyl-labeled glycerate. The carboxyl-label of
glycerate in corn was 20 times that of the a and B carbons.
while in the other plants it was only 2 to 5 times. However.
the glycerate and serine were not as carboxyl-labeled as
B-P-glycerate. and it was suggested that there were two
sources of glycerate, one from BAP-glycerate by phosphatase
action and the second coming from the glycolate pathway.

At times of 60 seconds or*greater. serine became uniformly
labeled, while glycerate remained carboxyl-labeled, thus
indicating serine formation from the glycolate pathway was
dominant.

Hess and Talbert (29) found considerable glycerate
(#-101 of total 1“002) formed during 14002 experiments with
tobacco. The glycerate and BAP-glycerate were carboxyl-
labeled through 30 seconds with the same percent distribution

31

in the carbons of both. At the same times glycolate and
glycine were uniformly labeled. Serine was slightly car-
boxyl-labeled at h and 11 seconds and was uniformly labeled
by 30 seconds. The specific activity of the 1“c in the
carbon.atoms of glycerate was much less than that of 3eP-
glycerate or glycolate. Such results would possibly indi-
cate a large reservoir of glycerate. The equilibration of
the 1"‘C in glycerate was as rapid as in BAP-glycerate indi-
cating a very active reservoir of glycerate. Had the
glycerate been an artifact of the killing procedure the
ratio of the amount of free glycerate to BAP-glycerate
would be constant. but this definitely was not the case
with tobacco (29). Thus it would appear that most of the
glycerate was the result °f.l£m!$!2 enzymatic hydrolysis
of 3-P-g1ycerate.

Bidwelleggwgl. (91) using chloroplasts from the
large marine alga Acetabularia, found that glycerate was
labeled after about 3 minutes of 1“002 photosynthesis. At
3 minutes the 1“c incorporation into 3-P-g1ycerate was
about half maximal. while incorporation into glycolate was
about 1/3 maximal. The glycerate pool was 1/8 the glycolate
and glycine and serine pools. and 1/6 the BAP-glycerate pool.
After the pools were saturated and the lights were removed.
there was an immediate Pvis sec) 80% increase in.3-P-glycer-
ate. which then returned to a steady state level 20% above
the predarkness level. Glycerate in turn increased 60%
within 2 minutes of darkness followed by a very slow rate

32
of decline. At the peak the radioactivity of the glycerate
pool was 5% of the total soluble luC. The investigators
were unable to explain the glycerate formation. Only 2%
of the total 140 was outside of the chloroplast.

The amount of glycerate in leaf tissue is quite
significant. Palmer (92) reports that 0.5 to 1.5% of the
dry weight of tobacco leaves is glycerate. Bruin (7“)
found 10-28% of the total 1"002 fixed by the Cu-plants
pigweed, corn and sugarcane during steady state photosyn-
thesis experiments to be in glycerate.

In summary. glycerate is an early oarboxyl-labeled
compound in 1“002 photosynthesis experiments in all plants
studied. The glycerate pool is significant and highly
active. The kinetics of glycerate formation and labeling
indicate that it is formed by quickly hydrolyzing BAP-
glycerate. Without considering a phosphatase, the action
of known.metabolic sequences and data cannot account for

all the glycerate formation in plants, however very little
data is available for Chaplants.

Acid Phesphatases from Leaves

Acid phosphatases from a number of plant tissues have
been reported (93-113) but in only a few cases have the
studies involved green leaf tissue (23, 93, 99. 102. 106.
112). Very few investigations present evidence for Speci-
ficity or sufficient purification to indicate whether or

not one enzyme was responsible for the activity observed.

33

This review will be limited to acid phoSphatase of green
leaf tissue. .

The only specific acid phosphatase from leaves that
has been reported thus far is P-glycolate phosphatase (23,
2h, 76). It is ubiquitously distributed in green leaves
(this thesis) and has been isolated from spinach (76),
tobacco (23) and green.algae (Randall and'Tolbert, unpub-
lished results). The enzyme from tobacco was shown to be
specific for its substrate with Optimal activity at pH 6.3.
but under some conditions divalent cations lowered the
optimum to about pH 5. The enzyme required a divalent
cation with Zn++. Mg”. and Co++ providing the highest
activity and EDTA completely inhibited the enzyme. Citrate,
isocitrate or cis—aconitate at 10'3 H were required for
enzyme stability (11h). Tim enzyme showed typical phospha-
tase inhibition by fluoride, but no inhibition by 14+)tar-
trate. Inhibition by cysteine, glutathione and p—chloro-
mecuribenzoate indicated a sensitive sulfhydryl or disulfide
bond on the enzymes. P-Glycolate phosphatase was formed
during bicgenesis of the chloroplast (76) and was localized
on or in the chloroplasts of C3-plants (70. 76). It has
been proposed that the enzyme functions as a part of a
permease or gives direction to glycolate excretion from the
chloroplast. P-Glycolate phosphatase may be tm link
between the site of glycolate biosynthesis (chloroplasts)
and the site of glycolate metabolism (peroxisomes), thus
performing a very specific function (115).

34

Another acid phosphatase(s) requiring divalent cations
has been reported by Boroughs (93), who found that at least
two phosphatases (two pH optimums) which hydrolyzede-nitro-
phenylphosphate were present in leaves of sugar beet and
tobacco. All of this phosphatase activity could be elimin-
ated by acid dialysis or addition of chelators. The enzyme
activity could be restored most effectively by addition of
Cu*+. '

Aside from the P-glycolate phosphatase the most
extensive study on an acid phosphatase in leaf tissue has
been presented by Shaw (112) on a BOO-fold purified, tobacco
leaf enzyme. Optimal activity was at pH 5.6, it did not
require divalent cations. was unaffected by sulfhydryl rea-
gents and was located in the cytoplasm. Fluoride, arsenate,
molybdate, and phosphate were inhibitors. The enzyme was
not very specific, for it had equal activity towards, ATP.
AII’. PP 1. NADP. 3-P-glycerate, 2-P-glycerate, PEP. phenyl
phosphate and‘p-nitrophenylphosphate. The enzyme was slow
to hydrolyze 5'-nucleotides in comparison to 3'-nuc1eotides,
and it did not exhibit diesterase activity towards RNA or
DNA. The carbohydrate esters were hydrolyzed quite rapidly
(about 2/3 of the rate for.NTP) and nonpspecifically. The
relative specificity was fairly unchanged during the purifi-
cation.

Roberts has reported on wheat leaf acid phosphatases
in a series of investigations (96-98, 101-1010 and concluded
that there could be as many as eleven phosphatases based on

35
various levels of inhibition by 17 inhibitors. A typical
example was a B-glycerol acid ph08phatase, requiring no
cations and operating optimally at pH 5.7. The phospha-
tases were reported as being non-specific, but no data were
presented with regard to the various substrates. The
second type of leaf phosphatase that Roberts reported was
a 5'-nucleotidase, which also possessed phosphotransferase
activity (104). No characterizations of the enzymes were
presented which would indicate function or location. Again
most of the studies were performed with‘p-nitrophenylphos-
phate as substrate, which is rapidly hydrolyzed by most
phosphatase except P-glycolate phoSphatase.

Plant seedlings have much higher acid phosphatase
levels than fully developed plants or leaves. An acid
phosphatase from seedlings of dwarf beans has been reported
(106) which was relatively non-specific (hydrolysis of 3-P-
glycerate, FdiP. and glucose-iéP was 1/10-1/8 that of p-
nitrophenylphosphate), did not require cations, and had a
pH Optimum of 5.3 to 6.3. The enzyme hydrolyzed 3'-nucleo-
tides but had little activity towards 5'-nucleotides and
had no diesterase activity. An.acid phosphatase purified
lOOO-fold from lupine seedlings (99) has also been reported.
{This phosphatase had pyrophosphatase and monoesterase activ-
ity, but did not hydrolyze 5'-nucleotides, polymetaphoSphates
or nucleic acids. The rate of hydrolysis of sugar phosphates
was very nonpspecific and almost as high as for‘p-nitrophenyl
phosphate. It was inhibited by CW. 2n++. and Co“ and was

36
slightly stimulated by Mn‘H' and Mg”.

Pierpont (97, 98) isolated an acid phoSphatase from
pea seedlings which was very nonspecific, hydrolyzing poly-
and metaphosphates, nucleic acids, nucleotides and other
monoesters. The enzyme activity was optimal between pH 5
to 6, and required no cations and showed typical fluoride
and molybdate inhibition.

Acid pyrophosphatase from Spinach leaves which would
hydrolyze PP1. ATP. ADP but few sugar phosphates was
reported by Forti (107). but no function for this enzyme
was proposed. Fortilgt a}. (105) also isolated and charac-
terized a nucleotidase from pea leaves which hydrolyzed 3'-
and 5'-nucleotides and pyrophosphate linkages. This enzyme
hydrolyzed PEP as rapidly as p-nitrophenylphosphate but was
very inactive toward B-glycerolphosphate, glucose-64F and
ZAP-glycerate. The enzyme very actively converted NAIP(H)
to NAD(H), and speculation exists for this hydrolysis as a
regulator mechanism. The enzyme activity was optimal at
pH 5.9. required no cations (stimulated by EDTA), and
possessed a Km of 3 x 10-“ M for ATP and 9 x 10"5 M for PEP.

A perusal of the literature on acid phosphatases in
leaves leads me to conclude that there are few generalities
about the acid phosphatases. Thus far. most of the studies
have been quite incomplete, but indicative of several differ-
ent enzymes. Most have pH optimum between 5.0 and 5.9 and
are nonPSpecific with respect to substrate. P~Glycolate

phosphatase is an exception. It is substrate specific and

37
has a pH optimum of 6.3. A divalent cation requirement for
some. but not all, of the leaf phoSphatase has been shown.
This thesis adds another acid phosphatase to the list of
those isolated from plant leaves, and the studies indicate
it to be different from those reviewed above.

MATERIALS AND METHODS

Plants

Sugarcane, Saccharium officiarum (variety C.I. u1-223)
was grown continuously in the greenhouse from stem nodes
which were obtained originally from L. P. Hubert of the
United States Sugarcane Field Station, Canal Point, Florida.
leaf tissue for enzyme purification.was from.plants 10 to 15
feet in height and 3 to 18 months old. New plants were
propagated regularly from stem nodes. The sugarcane and
other plants were grown in regular greenhouse mix with
additional sand and were treated with Hoagland's nutrient
solution on a regular basis. Etiolated tissue was grown in
controlled environment chambers in.vermiculate and Hoagland's
nutrient solution.

The plants used in the survey investigations are
listed in.Table XVI. Most of these plants were grown in the
greenhouse and were 3 to 6 weeks old when leaves were taken
for experiments. Other plants were obtained from specific
cultures or gardens on campus. Chlorella pyrenoidosa (Warburg
strain) was grown as described by Hess (116). Cultures of
Chlamydomonas reinhardtii were from E. B. Nelson. The
extracts of aquatic plants (Elodea'ggggg and Sagittaria)
were donated by R. Donaldson.

The leaf tissue from soybean.(Glzcine‘m§§ L. Merrill)

38

39
varieties Chippewa,, Wayne, Hark, Harvasoy. Grant, Amsoy,
Clark 63, Corsoy. Richland, Scott, Kent, Lee, Kanrich,
Hawkeye, and Seneca, were obtained from.the experimental
plots of the USE! Regional Soybean.Research Laboratory at
Urbana. Illinois.
Light intensity was measured in foot candles with a

Weston.hodel 756 Illumination Meter.

Chemicals

The chemicals and biochemicals used during the
research for this thesis were reagent grade and for the
most part the most pure available. Heavy metal salts of any
of the biochemicals were exchanged to appropriate counter
ion before use.

Aldrich Chemical Co. Nutritional Biochemicals

 

 

Corp.

ChromotrOpic acid

8 -A lanine
cc-Dipyridyl

Citric acid
8-Hydroxyquinoline-5-

sulfonic acid DL—Serine

8-Hydroxyquinoline Sodium azide
ZéPyridylhydroxymethane Hydroxypyruvate (L1)

sulfonic acid
Dihydroxytartaric acid
Dihydroxymaleic acid
Isonicotinyl hydrazide
0- .m-Phenanthroline

Worthington
Catalase
Bquylase

Glutathione
Carbamyl phosphate
5'-Citydylic (Na)
5'-Guanylatic (Na)
Sodium/Pyruvate
‘Phosphoeerine
5'-Uridylate (Na)
Oxalic acid

#0
General Biochemical Co.
Glycolic acid
P-Glycolic acid

Calbiochem
IpAepartate
LnAlanine
L-Cysteine

Malic acid
Malonic acid
Phytic acid
Thioglycolate
mesoATartaric acid
14+)Tartaric acid
L(-)Tartaric acid

DL-G lyceralde hyde-3..
phosphate

PhOSphohydroxypyruvate
Phosphochloine
Oxaloacetic acid

G lyc o lalde hyde
phosphate

Creatine phosphate
ZAP-Glycerate
Fructose-6-phosphate
ciseAconitic acid
Iso-Citric acid
DL-c-Bydroxybutyrate

Sigma
DL-G lyce rate

IpAscorbic acid
Cacodylic acid

peChloromercurobenzoic
acid

Glycylglycine
Dihydroxyacetone ph08phate
Trizma base

Glyoxylate

NAD, NADP. NAIPH, NADH
Phosphoenol pyruvate
Pyridoxa1-5'-phosphate
Dithioethreitol

bisefi-Nitrophenyl
p osphate

peNitrophenyl phosphate
Bovine serum albumin

Phenolphathalein
diphOSphate

c- and B-Glycerol
phOSphate

Glucose-6-phosphate
Fructose-1,6-diphosphate
Ribulose-1.5-diphosphate
Ribose-S-phosphate

AMP. ADP. ATP

41

Eastman.0rganic Chemicals Merk

 

(Amino-oxy)acetic acid Barbital
Diethyldithiocarbamic

acid General Aniline and Film
Iodoacetic acid Polyclar AT
‘p-Methylaminophenol

sulfate

Protein1 Chlorophyll and Reducing Sugar

The method of Lowry'gtflgl.. a modified Folianiocaltau
method (117), was used whenever sufficient protein sample
was available. Lowry proteins on a semi-microscale were run
on samples of 0.10 ml with 1.0 ml Lowry reagent C and 0.10 ml
FolinAPhenol reagent. The phenol reagent used was a 1:1
dilution.with H20 of 2.0 N Phenol Reagent (Fisher Scientific
Co.. Fairhaven. N. J.). The absorbance of protein reaction
was measured at 660 nm. The protein standard was crystal-
line bovine serum albumin.

For determining protein content of samples when loss
of enzyme was undesirable, the 260:280 ratio method was used
with the formula (118):

mg protein X ml‘1 = 1.45 (A280) - 0.71“ (A260)

The absorbance at 280 nm on.Beckman.Model DU with a
1.0 cm light path was used for obtaining protein profiles of

column effluents.

ChlorOphyll determinations were by the procedure of
Arnon (119).

1+2
Determination of reducing sugar was by the method of
Nelson (120).

Phosphate Determinations

Method A: The standard determination of inorganic
phosphate was a modified method of Fieke and Subbarow (121).
To a 1.0 ml aqueous phosphate solution ideally containing 3
to 30 us Phosphorous was added 8.0 ml molybdate reagent
(50 ml 10 N N280), plus 100 ml 2.5% (w/v) (N31,)6ho7024-4hzo
plus 650 ml 320) and 1.0 ml elon reducing agent (3 g of
Na3803 plus 1 g‘p-methylaminOphenol sulfate, "elon," in 100
ml H20) (11h). After 20 minutes at room temperature the
absorbance was measured at 660 nm cn.a Coleman Jr. Spectro-
photometer. A standard curve was made using 0 to 40 ug P
as KHzPou per ml (Fischer certified reagent).

Method B: Inorganic phosphate was also determined by
an iso-butanol-benzene extraction procedure (11“). To a 2.0
ml aliquot of solution, containing no more than 8 ug of
phosphorous, 0.5 m1 of 6% (w/v) (NRu)6M07024-4H20 in.2 N
3280“ was added. The mixture was vortexed for 5 sec and
allowed to stand for 5 minutes. To this solution was added
2.5 ml iso-butanol-benzene (1:1 v/v) and the mixture vortexed
for #5 seconds. After the phases had separated. the absorbance
at 310 nm of a 1.0 ml aliquot from the upper phase plus 10 ul
absolute alcohol to dissolve the remaining 820 was read in a

Beckman Model H] and compared with a standard curve.

#3
Method C: A microphosphate determination (122) was

 

also used which was 8 times more sensitive than the method
of Fiske and Subbarow (Method A). To 0.30 ml of solution
with less than.0.0h umole phosphate was added 0.7 ml
ascorbic acid-ammonium molybdate reagent (1 part 10%
ascorbic acid. plus 6 parts 0.h2% (w/v) (NHu)6M°702u'uHZO in
1 N H280“). After a 20 minute incubation at 45° the

absorbance at 820 nm was measured with.a Beckman.Model DU.

glycerate Determinations

Glycerate was determined by the method of Bartlett
(123). To a 0.2 ml sample containing between 0.01 to 0.30
umoles glycerate was added 5.8 ml 0.01% chromatropic acid
(10 mg recrystallized #,5-dihydrcxy, 2,7-naphthalene-disul-
fonic acid per 100 ml concentrated sulfuric acid). The
reaction mixture was mixed thoroughly and placed in boiling
water for exactly 30 minutes and then cooled abruptly in an
ice bath to room temperature. The absorbance at 695 nm
was measured with a Beckman Model DU and compared against a

standard curve.

Enzyme Assays
Standard assay for 3-P-glycerate phosphatase: A

reaction solution containing 12.5 umoles BAP-glycerate,
100 umoles sodium cacodylate buffer, pH 5.9. and enough
H20 to bring the final volume to 0.75 ml was equilibrated
to 30° in a conical test tube. The reaction was initiated

with enzyme unless otherwise stated. The enzyme reaction

44
was run for 10 minutes and was terminated by the addition of
0.25 ml of 10% (w/v) trichloroacetic acid (TCA). For crude
or low purity enzyme, the denatured protein was removed by
centrifugation. To determine the amount of phosphate hydro-
lyzed from the substrate, phosphate determinations were per-
formed on the TCA treated reaction mixture (0.5 ml aliquots
for crude or low purity enzymes). Reagents for the modified
Fiske and Subbarow phosphate (Method A) were added: 8.0 ml
acid molybdate solutions and 1.0 ml elon reducing solution
(4.0 ml and 0.5 ml respectively for 0.5 ml phosphate solu-
tions). Control or zero time assays were done by adding the
0.25 ml 10% TCA to the 0.75 ml reaction mixture before the
enzyme was added.

P-Glycolate phosphatase (PhoSphoglycolateJhospho-
hydrolase E.C. 2.1.2.18): This enzyme was assayed by the
method of Anderson and Tolbert (24). The reaction mixture
contained 0.50 ml of 0.2 M sodium cacodylate buffer, pH 6.3.
and 1 mM MgSOu. 0.25 ml of 20 mM P-glycolate, and after
equilibration to 30°. the assay was initiated with enzyme
for a 10 minute reaction period and terminated with 0.25 ml
of 10% trichloroacetic acid.

One unit of enzyme is defined as the amount which
catalyzes the hydrolysis of 1 umole substrate per minute
under conditions of the standard 3AP-glycerate phosphatase
assay. Specific activity is defined as units of enzyme per
mg of protein.

Glycolate oxidase (E.C. 1.1.3.1). catalaee (E.C.

45
1.11.1.6), and NADR-hydroxypyruvate reductase (E.C. 1.1.1.29)
were assayed spectrophotometrically as described by Tolbert
53 3;. (25).

Substrates

The BAP-glycerate was obtained as the barium salt-
dihydrate from.C. F. Boehringer and Soehne, GmBH. Mannheim.
West Germany. The barium salt was converted to free acid by
passage through 200-400 mesh Dowex resin (BioRad AG-50W—X12)
in the hydrogen form and then adjusted to pH 5.9 with base.
Paper chromatography revealed no other organic phoSphate
esters or organic acids. About 1.6 percent of the phosphate
present was as inorganic phosphate.

P-Glycclate was obtained as the tricyclohexylammonium
salt from General Biochemicals and was converted to the free
acid by passage through Dowex resin (BioRad AG-50W-12) in
the hydrogen form and adjusted to pH 6.3 with base. Previ-
ously, Dr. D. E. Anderson (114) had established the purity
of this substrate. In general, fresh solutions were pre-
pared weekly and stored at -18° to minimize endogenous
phosphate resulting from non-enzymatic hydrolysis.

All other substrates used were the most pure available

and are listed in Chemicals section.

Isoelectric Focusing
The apparatus was an Electrofocusing Column type IKB
8100* having a capacity of 110 ml. A density gradient of

 

*The author gratefully acknowledges the use of the
eéeitrofocusing equipment of John E. Wilson and his helpful
a v ce.

46
sucrose, in.which.Ampholine. carrier ampholytes, in the pH
range from 3 to 10 were dissolved, was made by successive
dilution of twenty-four 4.6 ml aliquots of 47 percent (w/v)
sucrose-water-ampholyte (1.25%) solution with a water-
ampholyte (0.4%) solutiticn. These aliquots were layered
on 22 ml of cathode solution containing 200 ul ethanolamine
in 55 percent (w/v) sucrose. The gradient column was comp
pleted by layering 5 to 7 ml of anode solution (1.9% sul-
furic acid) on the top of the gradient.

The enzyme was introduced into the column in one of
the middle aliquots of the gradient in place of the non-
eucrose containing portion. The column was operated at a
potential of 300 V for 36-65 hours at 4°. The column was
drained by gravity with a flow rate of 60 to 90 ml per hour.
About 2 ml fractions were collected. The pH of the fractions
was determined with a combination electrode on a Leeds and

Northrup pH Meter.

Gel.Filtration

Sephadex G-200 (40-120 u) from Pharmacia was routinely
prepared for chromatography as follows. The dry gel was sus-
pended in water and swollen on a steambath for 5 hours.
The suspended gel was allowed to settle for 9 minutes and
the suspended material remaining in the supernatant was
decanted. This fining process was repeated 10 times before
the gel was equilibrated with 0.02 M sodium cacodylate buffer,
pH 6.3 and 1 mM EDTA at 4°. Before pouring a column the gel

4?
was degassed for 10 minutes using a water-aspirator system.
The columns were poured and operated with the same head
height of 10 cm. Most columns were 2.6 x 46 cm with a 2 cm
pad of 0-25 Sephadex (prepared in identical manner) on the
top. The flow was descending and controlled by a mariotte
flask. All molecular sieving gels used in this work were

prepared in a similar manner.

Ion.Exchange Chromatography

Phosphocellulose (cation exchange resin. 1.24 meq/g)
from Serva (Gallard-Schleisinger) was suspended in H20.
allowed to settle 20 minutes, and the remaining suSpended
material removed by decantation. This fining process was
repeated 10 times. The resin was then washed with 0.1 N KOH
and 320 until the XOR wash was colorless. The resin was
finally equilibrated with 0.05 M sodium acetate buffer,
pH 4.5. at 4°. The 1.4 x 10 cm columns were poured and
washed with 50 volumes of the 0.05 M sodium acetate buffer,
pH 4.5.

DEAE-Cellulose DE-52 (Anion exchanger 1.0 meq/g)
from.whatman.was prepared in a manner similar to the P-cellu-
lose ion exchanger.

Carboxymethyl-Sephadex (cation exchange gel. 4.5
meq/g), G-50 medium grade, from Pharmacia was suspended in
HZO and swollen 1 hour on a steam bath. The gel was allowed
to settle for 8 minutes and then the remaining suspended

material removed by decantation. This process of fining

48
was repeated 10 times before the gel was equilibrated with
10 volumes of sodium acetate buffer, 0.05 M. pH 5.0. After
the 1.4 x 10 cm.columns were poured, the column was washed
with 50 volumes of the acetate buffer before application of
enzyme.

Profiles of the gradients were determined by adding
either DCPIP or Blue Dextran 2000 to the high-salt chamber
of the gradient forming apparatus. Absorbance at 600 nm
was determined on fractions of a calibration run. and all
gradients used were found to be linear.

Dialysis tubing. a product of Union Carbide Co.. was
heated on a steam bath in 10 mM EDTA for 8 hours, with fre-
quent changing of the EDTA solution. The tubing was then
washed extensively with deionized, distilled water and
stored at 4° in 20% ethanol.

Paper Chromatography

Paper chromatography on Whatman No. 1 filter paper
was run on samples that were spotted on the origin and dried
with warm air from a hair dryer. Chromatograms were pre-
equilibrated with solvent and chromatographic development
was done at room.temperature. The solvent system of‘n-butanol-
propionic acid-water (4) was prepared from equal volumes of
solutions A and B (A: 1246 ml of'npbutanol plus 84 ml H20;
B: 620 ml propionic acid plus 790 ml H20). A basic solvent
system.used was iso-butanol-95% ethanol-water-diethyl amine
(80:10:20:l) (116).

49

Phosphates on.paper chromatograms were detected by
the molybdate spray and UV light treatment method of Bandurski,
and Axelrod (124). Organic acids were detected by an indi-
cator. 0.04% (400 mg/l 95% ethanol) bromocresol green, in
the acid form for basic chromatograms and in the base form

for acidic chromatograms.

PreparatogyfiMethods

Chloroplast isolation from sugarcane leaves was by
the method of Baldry _e_t_:_ 2.}- (125). Cacodylate or MES buffer
was substituted for the pyrcphosphate buffer in the grinding
medium because of phosphate assays later. Subcellular
particles were separated by differential centrifugation, and
the isolation of peroxisomes from plant extracts followed
procedures developed in this laboratory (25, 26).

Isolation of chloroplasts from Spinach utilized a
sorbitol medium containing 0.33 M sorbitol. 0.01 M Na¢P207
buffer, pH 6.5, 5 mM MnClz, 2 mM MgC12. and 2 mM isoascorbate.
At 4°, washed and deribbed spinach leaves were homogenized
at high Speed for 8-10 seconds in a Waring Blendor. The
homogenate was squeezed through 8 layers of cheese cloth
and the resulting brei centrifuged at 4000 x g for 90 sec.
The pelleted chloroplasts were resuspended in 0.33 M
sorbitol. 1 mM MgClz. 1 mM MnClz. 2 mM EDTA and 50 mM Hopes
buffer pH 7.6. and recentrifuged to pellet the chlorOplasts.
This process was repeated as many times as necessary to
remove non-chloroplast enzymes. Glycolate oxidase was used

as a marker enzyme for non-chloroplast enzymes.

50

Sucrose density centrifugations for sedimentation
velocity determinations were run according to the procedure
of Martin and.Ames (126). Polyacrylamide gel electrophoresis
was performed according to the method of Orstein (127), Davis
(128), and Reisfeld 22.21- (129, 130). Running gels approx-
imately 7 cm long were used with samples layered directly
on the gels. A current of 5 milliamps per gel was applied.
Electrophoresis at pH 4.3 was in B-alanine-glacial acetic
acid buffer (130). Electrophoresis at pH 7.5 was in a
barbitolpTris buffer system and at pH 8.9 1n.Tris-glycine
buffer (129).

SDSePolyacrylamide electrophoresis was run according
to the method of Shapiro g3; g_l_. (131) . with the help of
J. C. Johnson and Dr. J. A. Boezi of this department.
Samples of enzyme in a solution of 1% SDS, 1% 2-mercapo-
ethanol and 0.1 M pH 7.1 phosphate buffer were incubated at
37° for 3 hours. Samples of the denatured and reduced enzyme
were layered on 7 cm. 5% polyacrylamide gels and a current of
6-7 milliamps per gel was applied. Standard protein samples
of known molecular weight were treated identically. The
buffer system was 0.1 M. pH 7.1 phosphate with 1% SIB present.

RESULTS

PART A:
3-P-GIXCERNTE PHOSPHATASE FROM SUGARCANE LEAVES

I. Purification of Sugarcane ZAP-Glycerate Phosphatase

The term enzyme as used in the subsequent paragraphs
in synonymus with 3-P-glycerate phosphatase. Table II con-

tains a summary of the purification procedure.

Extraction.from Sugarcane Leaves

Leaves from sugarcane plants from the greenhouse were
harvested in mid-afternoon only on days with full sunlight
and, when possible, after a day with full sunlight. The
chosen leaves were dark green, fully expanded, approximately
100 cm in length and 5 to 7 cm wide. The leaves were washed,
deribbed, and diced into 4-5 cm pieces using a paper cutter.
All subsequent steps were done at O-4°. Sixty gram portions
of the diced leaves were immersed in 5 volumes of extraction
medium containing 20 mM sodium cacodylate buffer, pH 6.3
1 mM EDTA, 20 mM ascorbate and 2% (w/v) Polyclar AT (cross-
linked, high.molecular weight. polyvinyl pyrollidone). The
tissue was allowed to soak for 15 minutes and then was homog-
enized by a Haring Blender at high speed for two minutes.
The homogenate was poured into a wine press lined with 6

51

52

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cow 0.:: Fm om.o owwwm oowumooauomum ocouooc umufim
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Omawnm o.H OOH Hm.o owman uomuuwo ensue
Awav Aswe\eoaoaiv
cwououm choa uooaaouuom macaw & .uod .am mums: scauooum

 

 

*mo>ooq oecouowom w cow scum oecucoamosm oumuoomawumum we monocoawnuom can mo humsssm .HH canes

53
layers of cheesecloth. Extracts of 600-800 g of leaf tissue

were pooled quickly in the wine press and the brei squeezed

as dry as possible to yield the crude enzyme extract.

pH Fractionation

As rapidly as possible after the extraction, the pH
of the crude extract was adjusted to 4.5 with 1 N HCl and
centrifuged at 14,000 x g for 30 minutes. The precipitate
was discarded and the supernatant was readjusted to pH 6.3
with 1 N NaOH. The pH stability of the crude extract and
the Specific activities of the crude extract and the Specific
activities of the crude enzyme after treatment at the various

pH'S are shown in Figure 8.

First Acetone Fractionation

To the acid fractionated enzyme, a volume of reagent
grade acetone equal to 35% of the enzyme volume was added
through 4 polyethylene tubes (1 mM I.D.) at approximately
5 ml per minute. The solution was stirred continuously
and the acetone was kept at -5° by the use of a salt bath.
After the acetone had been added, the mixture was equilib-
rated by slowly stirring for an additional 30 minutes and
then centrifuged at 14.000 x g for 20 minutes. The precipi—
tate was discarded and to the supernatant was added acetone,
as before, equal to 35% of the starting enzyme volume. The
resulting suSpension was equilibrated for 30 minutes, cen-
trifuged at 14,000 x g for 20 minutes and the supernatant

was discarded. The precipitate was suSpended in a solution

54

of 20 mM sodium cacodylate buffer, pH 6.3. and 1 mM EULA
equal to 20% of the acid fractionated enzyme volume. The
use of a glass Potter-Elvejehm homogenizer was necessary
to adequately resuSpend the precipitate. The suSpension
was stirred for 2 hours to ensure that the enzyme was
dissolved completely and then centrifuged at 14,000 x g
for 30 minutes to remove the insoluble material. The

supernatant was designated the first acetone fraction.

Ammonium Sulfate Fractionation

To the first acetone fraction was added 33 g solid
(NH4)2804 per 100 m1 of first acetone fraction enzyme. The
solution was equilibrated by slowly stirring for 20 minutes
after the (N34)2304 had dissolved. The solution was centri-
fuged at 14.000 x g for 20 minutes and the precipitate dis-
carded. To the supernatant, 22 g more solid (NH4)2304 per
100 ml of the first acetone fraction was added, the solution
equilibrated and centrifuged as for the first precipitation.
The precipitate was dissolved in 20 mM sodium cacodylate
buffer pH 6.3 and 1 mM EDTA equal to 10% of the volume of
the first acetone fraction. This was termed the ammonium

sulfate fraction.

Second Acetone Fractionation

In a manner Similar to the first acetone fractionation,
the second acetone fractionation was carried out by addition
of acetone at -5° equal to 63% of the volume of the ammonium

sulfate fraction. The system was equilibrated, centrifuged

55
at 14,000 x g for 15 minutes and the precipitate was dis—

carded. TO the supernatant, another volume of cold acetone
equal to 47% of the volume of the ammonium sulfate fraction
was added. After at least 30 minutes of equilibration the
precipitate was collected by centrifugation at 10.000 x g
for 10 minutes and the supernatant discarded. The precipi-
tate was suSpended in 5 ml of 20 mM cacodylate buffer, pH
6.3 and 1 mM EDTA. The suSpension was centrifuged at 40,000
x g for 20 minutes to remove insoluble material, and the
resulting supernatant was designated the second acetone

fraction.

Sephadex 0-200 Gel Filtration

 

Solid sucrose was added to the second acetone fraction
to make it a 10% (w/v) sucrose solution. The enzyme-sucrose
solution was layered under the tOp buffer of a 2.5 x 46 cm
0-200 Sephadex Column Operating in a descending manner and
equilibrated with a solution of 20 mM cacodylate buffer, pH
6.3, and 1 mM ETTA. The column was eluted with the same
buffer with a flow rate of 20 ml per hour and fractions of
about 5.0 ml were collected. Figure 4 shows the 3-P-glycer-
ate phOSphatase activity and 280 nm absorbance profiles.

The fractions with improved Specific activity (hatched area)
were pooled to yield the 0.200 Sephadex enzyme preparation.

Chromatography on.PhOSphocellulose
The G-200 Sephadex fraction was dialyzed for 36 hours
against 40 volumes of 0.05 M sodium acetate buffer, pH 4.5.

56

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57

U”u 093V) mews

 

 

 

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58

and 1 mM EDTA. The dialyzed enZyme was then adsorbed on
1.4 x 10 cm phOSphocellulose ion exchange column which had
been equilibrated with 0.05 M acetate buffer. pH 4.5. The
flow rate of the column was about 1 ml per minute and 10 ml
fractions were collected. Following enzyme adsorption, the
column was washed with 240 ml of 0.05 M acetate buffer, and
then the column was eluted with a 500 m1 linear gradient
from 0 to 0.5 M NaCl in 0.05 M sodium acetate buffer, pH 4.5
(Figure 5). The fractions of higher phOSphatase Specific
activity were pooled (hatched area) to yield the phOSpho-

cellulose enzyme preparation.

Chromatography:on Carboxymethyl Sephadex

The enzyme preparation from phOSphocellulose chroma-
tography was dialyzed for 18 hours against 40 volumes of
0.05 M acetate buffer, pH 5.0. and 1 mM EDTA. The dialyzed
enzyme was adsorbed on a 1.4 x 10 cm CM-Sephadex column.
previously equilibrated with 0.05 M acetate buffer, pH 5.0.
The column was washed with 200 ml of buffer and eluted with
a 500 ml linear gradient 0 to 0.5 M NaCl in 0.05 M acetate
buffer, pH 5.0. The column was Operated with a flow rate
of 1 ml per minute and 10 ml fractions were collected. The
phOSphatase activity and 280 nm adsorbance profile are shown
in Figure 6. The pooled fractions of highest Specific
activity were dialyzed against 40 volumes of 0.02 M sodium
cacodylate buffer, pH 5.9. with 1 mM EDTA to yield the CM-
Sephadex, 3eP-glycerate phOSphatase preparation.

59

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60

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63

Concentration of Enzygg

The pooled enzyme fractions from CM—Sephadex column
were concentrated by vacuum dialysis. A dialysis bag was
attached to the stem of a 40 mm funnel by co-insertion
through a rubber stOpper that was the size of the neck of a
2 liter Buchner flask. The enzyme was continuously added
to the dialysis bag through the funnel. The vacuum was
drawn on the system by a Cenco Hyvac-2 pump until bubbles
were Observed in buffer that had been added to the bottom
of the flask. The flask was closed by a stopcock connection
between the pump and the flask and the system was held at 4°.
Enzyme was added as the level receded in the system and the
vacuum was renewed about every 3 to 4 hours. It was possible
to concentrate 100 ml of enzyme to 5 ml in 24 hours or 5 to
10 ml in about 90 minutes. The inside surfaces of the
dialysis tubing were rinsed with buffer to help reduce the
loss of enzyme. The concentrated enzyme was removed from
the sac by either pouring the enzyme out or using a long
tip pipette.

Discussion of the Purification Procedure

Grinding Procedures: The age of sugarcane leaves was
a factor to be considered only with regard to the breaking
of the leaf tissue. Initial efforts to purify the enzyme
utilized sugarcane leaves flown in from Canal.Point, Florida.
The leaves obtained in this manner were used 8-36 hours

after harvesting, but still resulted in yields of enzyme on

64

a per gram fresh weight basis that were comparable to plants
grown in the greenhouse. Older leaves, low on the stock,
were so woody, stiff, and dry that they could not be properly
homogenized by the equipment available. Even the young,
relatively tender leaves were difficult to break completely,
and it was necessary to grind small pieces of them for 2
minutes in the Waring Blender at high Speed to solubilize
about 80-90 percent of the total activity (Figure 7). With
shorter grinding periods very poor recoveries were Obtained.
This procedure extracted most of the P-glycerate phOSphatase
activity, which is mainly in the mesophyll cells, but not
only about half of the P-glycolate phOSphatase and glycolate
oxidase activity of the bundle Sheath cells, which could be
completely broken only by further grinding in a roller mill
(Figure 7). The significance of these results will be
elaborated in Append ix A.

The most satisfactory grinding procedure started with
60 g of diced sugarcane leaves, which had been soaked in the
grinding medium for 15 minutes. They were ground for 120
seconds in 350 ml of grinding medium (20 mM cacodylate, pH
6.3. and 1 mM EDTA, 20 mM ascorbate and 1% Polyclar AT) and
the homogenate was squeezed through 6 layers of cheese cloth.
In the large scale preparations this was repeated numerous
times. However, this step plus the pH 4.5 fractionation had
to be completed within one hour to prevent accumulation of
excess phenolic oxidation products which could not be removed

from subsequent enzyme fractions. Rapid performance of these

65

Figure 7. The Percentage of the Enzymes Solubilized as a
Function of Homogenization Time or Use of the
Roller Mill*

Triplicated samples (30 g) of leaves were extracted by
the Waring Blendor at high Speed in 150 m1 of 20 mM cacodylate
buffer, pH 6.3. and 1 mM EDTA. At the designated times an
aliquot was removed and filtered through 6 layers of cheese
cloth. After 120 see the homogenate was squeezed through
cheese cloth and the residue passed repeatedly through the
roller mill while being mixed with the same buffer. Total
enzyme activities, protein and chlorophyll were calculated
from the sum of the two extraction procedures.

e———e 3AP-glycerate phOSphatase

I—l P-glycolate phOSphatase

X---X ChlorOphyll

0---O Protein

AF——A. Glycolate oxidase

 

*A description and use of Roller Mill is found in
Appe nd 1x A e

by

Ate

% of Total‘ Activity

"/0 of Total Activity

IOO

ll
Sugar Cane H
80- e H .3
Chl h ”‘0 ,IX ll ..
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60-— 45°" " 5
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30 60 9O |20”Roller MIII

66

 

 

 

 

 
  

 

 

 

 

 

 

 

Waring Blendor (sec)

 
   

 

 

 

 

 

 

 

 

 

 

 

67
initial steps yielded a clear, almost colorless supernatant
devoid of chlorophyll after the pH fractionation, and subse-
quent precipitations were also almost colorless.

Other methods of breaking up the sugarcane leaves
were tested and found unsatisfactory. The mortar and pestle
was very limited in its capacity and exhaustingly slow. In
the large commercial Waring Blendor inadequate breakage
occurred as well as rapid heating of the extraction medium.
The use of a large Hobart meat grinder, although efficient
in macerating the leaf tissue, resulted in accelerated poly-
phenol oxidase activity, which interferred with subsequent
purification of the phoSphatase. When sugarcane leaves were
ground in the Habart grinder without buffer and the macerated
tissue then placed in extraction medium, the phenolic oxida-
tion was severe. As described above, soaking the leaf tissue
in the extraction medium before homogenization in the small
Waring Blendor resulted in almost complete extraction of
enzyme without heating the medium or allowing unnecessary
oxidation.

Since the phenolic oxidation hindered subsequent
purification of the phOSphatase, 2% Polyclar AT was added to
bind phenolic and phenolic like compounds so prevalent in
plants (132). Likewise ascorbate was added to reduce unneces-
sary oxidation. Since in preliminary eXperiments the enzyme
indicated no requirement for metal cations and was inhibited
by various divalent cations, EDTA was routinely added. The
EDTA also slowed the phenolic oxidations.

68

Acetone and ammonium sulfate fractionations: Complete
equilibration during the precipitating procedures always
resulted in improved yields. When considerable amounts of
phenolic material were in the starting enzyme solution, the
first acetone fractionation removed a large portion of them.
Although.P-glycerate phoSphatase was soluble after acetone
precipitation, the bulk of the insoluble sticky protein had
to be diSpersed by a glass homogenizer and prolonged stirring
times were used to ensure complete suspension.

In scaling up to 800 to 1000 g of leaves for the final
two large enzyme preparations, the yields from the first
acetone fractionation were around 50% as compared to 80-90%
in smaller runs. It is thought that a change in the precipi-
tation pattern occurred and that more acetone should have
been used to precipitate the enZyme.

Results from ammonium sulfate fractionation were often
inconsistent for reasons unknown. The purification was
either very good at this step or quite low with a parallel
poor recovery. The rate of addition of the (NHh)230u was
not critical, but complete equilibration after addition of
the salt always improved the results and yield by at least
a 2-fold purification.

The second acetone fractionation resulted in a 2 to 5
fold purification with good recovery and served to concentrate
the enzyme for the 6-200 Sephadex gel filtration. The pre-
cipitated enzyme was collected in one centrifugation and

then dissolved in a small volume of 20 mM cacodylate buffer,

69
pH 6.3. and 1 mm ETTA. Good recovery required careful and
complete resuSpension of the precipitate. The insoluble
material was removed by centrifuging at 40,000 x g.

Solid sucrose up to 10% (w/v) was added to the second
acetone fraction for efficient sample application to the
Sephadex gel column. The enzyme-sucrose solution was
layered on a 6-25 pad on tOp of the 0-200 Sephadex column
in order to prevent disruption of the G-200 gel by the
heavy solution. A longer column should have improved the
results. but ascending column equipment was unavailable at
the time. A Blue Dextran sample through the 0-200 Sephadex
column permitted estimation of the void volume for an approx-
imate estimate of molecular weights. The enzyme activity was
slightly retarded by the gel giving a ratio of the elution
volume to void volume (Ve/Vo) of 1.36 (Figure ii). The
fractions were pooled. as indicated. on the criteria of a
higher specific activity than the applied fraction. The
phosphatase was completely excluded from.G-100 Sephadex.

Ion exchange chromatography: It was at this point
in the purification that difficulties arose. Since the
enzyme from the gel filtration step was diluted. it was
felt that adsorption on an ion exchange column would be
desirable. DEAE-Cellulose or DEAE-Sephadex was picked as
the most likely exchanger. but this was a most unfortunate
choice. The 3-P-glycerate phosphatase activity gave a
different pattern for every DEAE column attempted. Some-

times it was adsorbed entirely and other times only partially.

70
The enzyme activity was eluted in 2.? peaks depending on
pH. type of gradient. or concentration of applied enzyme.
Rechromatography of that enzyme not adsorbed gave a pat-
tern similar to the original attempt--some unadscrbed and
the remaining eluting in several peaks. Rechromatography
of individual, separated peaks gave the original peak and
any that followed in the original column, but none preceed-
ing it. Because of the potential of DEAE exchanger. consid-
erable effort was expended in trying to perfect the process
and determine the causes of multiple peaking and adsorbing
properties.

In the pH range of 5.6 to 7.0, it was not possible
to elute the enzyme from DEAE-cellulose in a reproducible
manner. Below pH 5.6 increasing amounts of the enzyme came
through without sufficient purification. Above pH 7.0 one
peak was obtained but only 10-20% of the activity was
recovered with equally poor purification.

The multiplicity of the enzyme peaks from DEAE
columns suggested the possibility of a number of phoSphatases
or isoenzymes being present. Since specificity data was
inconclusive. an isoelectric focusing experiment was per-
formed in a pH range of 3 to 10. The isoelectric focusing
resulted in only one peak of enzyme activity (see Figure in,
Section II). Since the isoelectric point (pI) of the phos-
phatase was about 6. a plausible explanation for the multiple
peaking may be the running of the IEIE columns too close to

the pI. Any slight differences in extraneous material or

71
other proteins with the enzyme or bound to it could cause a
"different" enzyme to elute just by slight charge differ-
ences. At the higher pH'S C>7.0) the differences would
become negligible but the enzyme stability then became a
problem. At pH's less than 6.0. the enzyme became positively
charged and more of it came through the column in the void
volume. Therefore. further purification attempts were
directed towards cation exchangers.

Phosphocellulose chromatography: The phosphocellu-
lose cation exchanger was tried with the idea that the phos-
phate group on the cellulose might bind the enzyme quite
preferentially. A 3-fold purification was obtained (Figure
5 and Table I). The amount of buffer used to wash the
column had no effect on the elution pattern nor did the
type of gradient. For either a linear and concave gradient
the enzyme eluted at about 0.2 M NaCl.

CM-Sephadex chromatography: The protein profile from
the phosphocellulcse elution suggested that a second cation
exchange column could give further purification. The CM-
Sephadex cation exchange column proved to be most useful at
pH 5.0 from which the enzyme eluted at about 0.25 M NaCl
(Figure 6). The fact that very little 280 nm absorbance was
found anywhere on the column made protein determinations very
difficult. and probably greater in error. For more accurate
determination of protein and the fact that enzymes are fre-
quently unstable when very dilute, the enzyme was concen-

trated by vacuum dialysis before characterization. Concen-

72
tration of the enzyme also resulted in increased Specific
activity (Table II).

Some other purification procedures which were tried
and proved unsuccessful in addition to the DEAE-cellulose
were: TEAE-cellulose: positive or negative absorption on
alumina CY gel: heat fractionation: ethanol precipitation;
and calcium phOSphate gel.

Stability: In a crude extract of sugarcane leaves
BAP—glycerate phoSphatase was stable at no for about a week
before microbial growth destroyed the preparation. The
activity in crude leaf extracts was completely stable to
-18° for at least 6 months. A reason for not storing the
enzyme in the crude extract was the slow continuous oxida-
tion of phenolic compounds which interferred with subsequent
purification of the enzyme.

The pH stability curve for the crude extraction of
3sP-glycerate phosphatase and the 0-200 (Figure 8qA) indi-
cates that it was unstable to the high pH and quite stable
to low pH. The stability of the enzyme at low pH was util-
ized for the acid fractionation step in purification. The
Specific activity of the enzyme at the various pH'S after
removal of precipitated protein is also shown in.Figure 8-A.
The pH stability was unaffected by increasing ionic strength.
However. enzyme from.G-200 Sephadex fractionation was rapidly
inactivated by (NHu)2304 and (N34)280u precipitation around
pH 6.0.

Partially purified enzyme was completely stable at

73

Figure 8. The Stability of BAP-Glycerate Phosphatase as
Function of pH and Ionic Strength

A. Aliquots of 20 ml of crude enzyme were adjusted to
indicated pH with 1 N NaOH or 1 N H01. The volumes were then
adjusted to 25 ml and incubated at no for u hours. Aliquots
of the samples were assayed and compared with controls (0———O).
The crude enzyme at the various pH's was then centrifuged and
adjusted to pH 6.3 with 1 N HCl or 1 N NaOH and their volumes
equalized by adding 20 mM cacodylate buffer. pH 6.3 and 1 mM
EDTA.

Aliquots of 100 ul of the Sephadex G-200 fraction
of 3-P-glycerate phosphatase were diluted with 0.9 ml of 0.1
M cacodylate plus 0.1 M acetate buffer or 0.1 M cacodylate plus
0.1 M glycylglycine buffers at indicated pH. After 4 hours
incubation at U° the samples were assayed and relative 3-P-
glycerate phosphatase activities were determined (A—A).

B. Aliquots of 50 ul of G-200 Sephadex fraction at the
various pH's were diluted with NaCl or sodium acetate solu-
tions of ionic strengths of 0.4 M (A). 0.6 M (0). 0.8 M (I),
and 1.0 M (X) and assayed after 4 hours.

 

 

 

 

 

 

 

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pH 5.9 and b0 for several weeks or as long as it was kept
sterile. Less than 5% of the activity was lost after stor-
ing at -18° for up to 3 months. The most purified enzyme
preparations were stable for at least 2 months at #0 at
concentrations so dilute the 280 nm absorbance readings

were negligible.
II. Biochemical Properties of 3-P-Glycerate Phosphatase

The Enzjme Assay

EnZymatic hydrolysis of 3-P-glycerate was linear over
the time period used and was also a linear function of the
enzyme concentration (Figure 9). The amount of enzyme used
in the enzyme assays was selected so that the phOSphate
released in a 10 minute period was between 0.2 umole and
1.2 umole. These conditions of linearity over the assay
time and enzyme concentration were checked frequently and at

each purification step of the enzyme isolation.

pH Optimum

The pH activity curves for BAP-glycerate phoSphatase
are in Figure 10. These curves were determined using the
highly purified enzyme after CM-Sephadex fractionation. The
pH optimum in the 5 buffering systems was between 5.7 and
6.0. When the activity values from the different buffer
systems were normalized to their highest value and then
averaged, the optimum pH was 5.9. The addition of the
divalent cation, Mg++ did not change the pH Optimum in any

76

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79
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way. The pH curves in the different buffers were quite
narrow and did not show distinct characteristics of either
acid or alkaline phoSphatases. The pH cptimum is not in
the range of most chloroplast enzymes (pH 7 to 8.5).

The pH activity curves for less pure enzyme were
shifted slightly to hhgher pH values. The enzyme had a
pH optimum of 6.3 in early stages of purification (Figure
11) through the second acetone fractionation. Consequently
pH 6.3 was used for assays of less pure preparations and is

possibly closer to the optimum'ig.vivo.

Kinetic Characteristics

The apparent Km of BAP-glycerate phosphatase from
sugarcane leaves purified through CM-Sephadex fractionation
for substrate ranged from 2 to 5 x 10'“ M from several
different enzyme preparations (Figure 12). The average
apparent Km was 2.85 x 10‘“ M. Considering the somewhat
non-specific nature of the enzyme, it should be emphasized
that there was no indication in the Lineweaver-Burk plots

of two enzymes acting on the substrate.

Effect of Temperature

The enzyme in sugarcane leaf homogenates was 50%
inactivated by heating to 50° for 3 minutes and about 80%
inactivated by 60° (Figure 13-A). The effect of tempera-
ture on the reaction rate with the purified BAP-glycerate
phosphatase is shown in Figure 13-B. In,a parallel experi-
ment aliquots (5 ul) of the enzyme at #0 was added to assay

700 —

0 4+
600 I- 3- PGA - Mg

500 ‘-

Units

400 P-

Relative

300i—

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P-Glycolote I O

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I K
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3 4 5 6 8 9

PH

h—
P
—

Figure 11. The pH Activity Curve for B-P-Glycerate Phospha-
tase and‘Paclycolate Phosphatase Using First
Acetone Fractions

Phosphatase activity on.34P-glycerate and.P-glycolate
was determined at indicated pH's using buffer systems of 0.1 M
cacodylate plus 0.1 M acetate and 0.1 M cacodylate plus 0.1 M
glycylglycine. Phosphatase activity was determined in
presence and absence of 10"3 M MgClz.

82

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86
buffer and substrate at the desired temperature and the reac-
tion.run for 1 minute. The rate of BAP-glycerate hydrolysis
was maximal about h2°. The enzyme stability declines rapidly
after 50° but is quite stable at 30° where the enzyme was
assayed in the standard procedure. From the Arrhenius plot
(insert Figure 13) the activation energy was found to be
about 9.8 Kcal. The Q10 value for the enzyme activity in
the 15° to 35° range was about 1.7.

Isoelectric Point

The isoelectric point (pI) of 3-P-glycerate phoSpha-
tase after the CM-Sephadex step of purification was about
pH 6.8 (Figure in). Nearly an identical profile was
obtained with the enzyme obtained from the 0-200 gel fil-
tration step, except that the pI was near to pH 6.5 (data
not shown). About 60% of either enzyme preparation that
was applied to the column was found in the peak, and 20-25%
of the activity trailed the peak in the fractions towards
the acidic side in the elution profile. The reason or sig-
nificance for the long trailing pattern is not known. It
is possible that some of the enzyme adhered to the sides of
the column or that the manner of draining the column did
not ensure even removal. The results are consistent with
the probability that only one 3AP-glycerate phosphatase was

present.

Effect of Ionic Strength
When the purified enzyme from the CM-Sephadex frac-

tionation was assayed in the presence of NaCl or sodium

87

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89
acetate there was a slight (12-15%) loss of activity above
0.3 M salt (Figure 15). When the enzyme was diluted 10-fold
at low ionic strengths G<0.05 M) in buffer at pH 5.9 and
held at room temperature for 5 hours before assaying about
30% of the activity was lost. However the enzyme was
perfectly stable to dilution and room temperature at an‘
ionic strength of 0.2 to 0.5 M (Figure 15). Prolonged
stability of the enzyme at u° with various ionic strengths
was excellent with essentially no loss of activity until

the pH was increased above 7.5 (Figure 8-A).

Substrate Specificity
The substrate specificity for 3-P-glycerate phOSpha-
tase was examined using hh substrates of which very few
(phosphoenol pyruvate and p—nitrophenylphoSphate) showed
activity greater than 50% of that for 3AP-glycerate hydrol-
ysis (Table III). For physiological reasons (Literature
Review) the functions of 3oP-glycerate phosphatase and
P-glycolate phosphatase may be related. P-Glycolate phos—
phatatase was Specific, but no absolute substrate specificity
was found for BAP-glycerate phosphatase. Since the purity
or homogenity of the enzyme has not yet been established,
one cannot state that the non-specificity is due to only
one phosphatase. However, the single peak in isoelectric
focusing would support only one enzyme. .Also the Lineweaver—
Burk plots indicate only one enzyme acting on BAP-glycerate.
During purification of the enzyme, the relative activ-
ity for BAP-glycerate hydrolysis increased through the

90

 

Control
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Sean

 

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m 0.2 w 0.3 0.4 0.5

Ionic Strength (NaCl or No Acetate)

 

 

Figure 15. The Effect of Ionic Strength on 3-P-Glycerate
PhOSphatase Activity

The Cid-Sephadex fraction was assayed in the presence of
either NaCl or sodium acetate of indicated ionic strength.
This standard assay was initiated by addition of a small
aliquot of enzyme (A—A). Also the activity in the CM-Sephadex
fraction was diluted 10 fold into 0.01 M sodium cacodylate
buffer, pH 5.9. containing NaCl or sodium acetate of indicated
ionic strengths, and allowed to stand 5 hours at room tempera-
ture before assaying at standard considions (0—_0).

Table III.

91

Specificity of 5-P-Glycerate Phosphatase

All substrates were present in 10 pmole amounts in the standard assay described

in Materials and Methods.

used (1 pl). Activities are shown relative to 5-P-glycerate at 100.

A

Enzyme purified through CHrSephsdex and concentrated was

 

Substrates Rel. Act. Substrates Rel. Act.
3-P-glycerate lOO Nicotinamide adenine 7
dinucleotide-P
DL-glyceraldehyde-j-P hl Nicotinamide adenine 11
dinucleotide-P reduced

2-P-glycerate 2h aePhenylphosphate h8
P-glycolate 11 biafpfiNitrophenylphosphate 0
P-glycoaldehyde .15 .2:Nitrophenylphosphate 66
Phosphoenolpyruvate 6h Phenophthalein diphosphate h8
Dihydroxyacetone phosphate 2h Pyridoxal-S-phosphate ll
a-Glycerol phosphate 1h Phosphocholine l
B-Glycerol phosphate 16 Glucose-6-phosphate 7
Phospholactate ll Glucose-l-phosphate O
Propansdiol phosphate 37 Fructose-6-phosphste 6
Phosphossrine 10 Pructosesl,6-diphosphste 51
Phosphohydroxypyruvate 17 Ribose-S-phosphate 7
Phosphoethanolamine 2 Ribulose-l,5-diphosphate ll
Adenosine triphosphate 57 6-P-Gluconate l7
Adenosine diphosphate 18 Carbsuyl phosphate 0
3'-Deoxysdanosine monOphosphate O Creatine phosphate 0
j'quenosine monophosphate 9 Pentaphosphata O
5'-Adenosine monophosphate 2 Decaphosphate O
S'-Uridine monophosphate 1h Pyrophosphate 9
5'-Guanosine triphosphate 26 Phytic acid 5
5'-Cytosine triphosphate 28

5'-Cytoaine monophosphate 5

 

92
ammonium sulfate fractionation step to 110-fold purification
(Table IV). However further purification of the phOSphatase
to 2530-fold did not significantly alter the ratio of activ-
ity toward the various substrates. Consequently it is
probable that one phosphatase is present which catalyzes the
hydrolysis of these various substrates. After the initial
steps of the purification, rates of hydrolysis of P-glycolate,
‘p-nitrophenylphoSphate, and pyrophosphate decreased and rates
of hydrolysis of ATP and fructose-1,6-dipho8phate increased
relative to 34P-glycerate. During one purification attempt
the specificity of 3-P-glycerate phosphatase was checked
immediately after the second acetone fractionation and then
rechecked after the enzyme had been stored in the coldroom
for 2 weeks and then for another 2 weeks at -18°. The rate
of hydrolysis of 34P-glycerate remained unchanged, but the
rate of hydrolysis of several other substrates had increased
for the enzyme stored at no and -18°. Thus there appeared
to be a loss of specificity with aging.

EDTA (1 mM) was used in the isolation procedure and
should inactivate divalent cation requiring phosphatases
such as P-glycolate phosphatase. The results in both Tables
III and IV were from assays without added Mg”, and thus the
activity of other phosphatases would be minimal. .Addition
of MgClz to the assays with purified P-glycerate phosphatase
did not alter the activity ratios for P-glycerate and the
other substrates, suggesting that the purified enzyme did

not contain other divalent cation requiring phosphatases.

93

.munoEnnoaxo wcnmsuom unnuuonoOon aonu oonooa osmunmt

 

 

no :0 I I I I I I ouo>snma Honoonauonm
I I mn I I I I I ouonaoona nonoozano
I I en I I I I I ouonaoona nonoomanm
we we a: :m am so mn mn ouonaaonano oaouusnm
on an I am mm mm mm mm onnnomonaaonm
3 mm mm mm. 6 mo mm mm 3 ssaaosa to «3832
I I I Am mm mm mm mm ouunaaonano onnaoooo4
I I mm ma 0n 0n n: on ouonauonaonmm
- - m m m o m m 3 Ba 3.1-0-3836
3 mm - .3 oo on ow : anowwnfiflfiwmfi
oo oo on an on ma. o9 own ooosaoosa .naosaononza
no am 2 we in a 2 S ousnoonnmInIm
: Z n s 3 3 om no mm 338.3»-.—
02 8 a 02 8a 02 03 OS 02 so on sonannIn
Ina xooonaom xooanaom , oncuou4 vomNAvnzv encuou4 nonuannuuanm oosno ousnuonom
no 8.0.6 saw a: ma

 

 

.anofinsnunounou I: On

an mononuonsm nuns homes omoumnaaona ouonoumanmIn onoonoum any menu: oomooas one: sounauansa uncano> one

neonuoonmnusm mo sowoum noonno> um autumnaoonm ouonoomnuImIm no aunonunooam o>nuonon .>H canoe

90

According to Thorn (133) and Dixon and Webb (13h) one
means of determining whether the activity toward two sub-
strates is due to one or two enzymes is to compare the
activity with such substrate Separately against the sub-
strates together. If two enzymes were present, the activ-
ities should be additive, and if one enzyme was present, the
activity would be the average of the two separately. When
this was done (Table V) the activity with 3aP-glycerate alone
was always greater than with BAP-glycerate plus a second
substrate. Thus the activities were not additive. but
neither were they the average of the two alone. Since the
rates were not additive for 3aP-glycerate with 11 other sub-
strates, one enzyme is suggested. A basic assumption for
this type of eXperiment is that the apparent Km's for the
two substrates should be about the same (13h). Although
the apparent Km's for the other phoSphate esters were not
determined, different values mhght well be expected and
probably could account for the varying results.

In other phases of this investigation we have been
concerned with activity ratio of P-glycolate phoSphatase to
BAP-glycerate phosphatase and with the physiological func-
tion of these two phOSphates. Consequently, the P-glycolate
phOSphatase activity in the first acetone fractionation of
the 3éP-g1ycerate phoSphatase was examined. This activity
did not have the properties of the Specific P-glycolate
phOSphatase (23, 2h), but rather it appeared to be a non-
Specific Mg++-dependent acid phoSphatase. The pH cptimum

95

Table V. Activity of 5-P-G1ycerate PhOSphatase with Two Substrates Persent

PhOSphatase activity was determined using the substrate alone

(10 umoles) and for 5-P-glycerate (10 umoles) plus a second phosphate
ester (10 umoles).

m

Activity (nmoles P released/10 min)

 

Ph08phate Ester

 

Alone + PGA Calculated*
Control, 5-P-g1ycerate 700 700 700
P-Glycolate 88 #76 59h
2-P-Glycera te 155 585 tau
a-Glycerol phOSphate 78 661 589
PhOSpholactate 58 595 579
PhOSphoserine 78 661 589
Phosphoethanolamine 15 662 559
PrOpanediol phOSphate 252 680 h76
G1ucose-6—phosphate 10 661 * 555
PhosPhocholine 78 6H2 589
Ribose-5-phosphate 97 718 599
Fructose-1,6-diph08phate 550 AMT 515

 

*The calculated value is based on the average of the activities of the
Substrates alone.

96
for P-glycolate hydrolysis was around pH 4 (Figure 12),
where as the Specific P-glycolate phOSphatase has a pH
cptimum of 6.3. The difference in rate of hydrolysis by
the first acetone preparation of the two substrates and

the pH optimum are emphasized in.Figure 12.

Effect of Metal Complexing Agents

Dialysis of BAP-glycerate phosphatase at each stage
of purification against #0 volumes 10"3 M EDTA at 9° for
48 hours did not cause any loss of activity. It was
initially concluded that the enZyme had no easily removable
metal cation, and EDTA was employed in the isolation proce-
dure for stability of the protein and for inactivating
other phoSphatases. In order to determine whether or not
the enzyme had a metal requirement, a number of metal com-
plexing or chelating agents were incubated with the enzyme
for 15 minutes under assay conditions and then the enzyme
reaction was initiated with substrate (Table VI). None of
the complexing agents, even at 10‘“2 and 10"3 M showed sig-
nificant inhibition of 3-P-glycerate phoSphatase that had
been purified 17h0-fold through the CM-Sephadex step of the

fractionation procedure.

Effect of Cations

The effects of various metal cations on 3-P-glycerate
phoSphatase activity are shown in Table VII. Enzyme (5 ul)
with a Specific activity of 562 purified through the

97

Table VI. The Effect of Metal Complexing Agents on 5-P-Glycerate
PhosPhatase Activity

The enzyme was from CM-Sephadex fractionation. The activities
were calculated relative to the control (no additives) at 100.

 

 

Final Concentration of Complexing Agent

Complexing Agent During Assay

 

 

10’2 M 10'8 M 10‘4 M

DithiothreitoI 109 101 102
EDIA 107 102 102
‘g-Phenanthroline * 95 10h
m-Phenanthroline * 100 100
8-Hydroxyquinoline 100 102 10h
8-Hydroxyquinoline-5-

sulfonic acid 102 100 100
oo-Dipyridyl * 96 106
Diethyldithiocarbamate 92 98 100
Sodium azide 99 101 100

 

*Formed a precipitate with reagents for determining phOSphate.

S98

 

mnuomvozz

 

 

 

 

 

 

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Inc
so no we -no 1++no mm we on mom ++on
Hen nan am -no +oaz am an em mom ++nz
OOH n0n oon Ioowosoa _+oz mm an m Ioosooo< ++sn
Inc moz
OOn oon mm mom +n om mm on -no ++oo
”no now
no mm a won an mm as am ooz so
Iousuuu4 .++ Inc ++
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++ ouauoo4 ++
no 2. a so a... -
++ Iououoo4
mm on n Ioooooo< as am no no -no as
-no ++ mom ++
en en a mom ++so 00n ecu OOH nonoaoo
z v-3 n 92 n «IS 2 38 x 0-2 2 «I3
sonsoo sonsao
anonuoo mo nonuonunousou dough anonuou mo canuenusoonoo Hanan
.mosua on» new ooas one: KooonaomIzu nwsonnu nonunnoa oanwno.mo AH: mv auosvnn4 .ouaonnawnu
an oouoSAubo on: ones noon .oowano>a anon acnuou nooo mo ounce osouns> scnw aonuw>nuos one .nouuso

manna onu an senuso onu nuns unease onu mo nonuensonn ounces ma 0 nouns ouonuan:a nuns oouonunSn was

nonuooon oseuso 0nu usnu uaouwo soonuoz one unannouax on oonnnuuoo as uso oonnnoo onus anonuooom

autumnaaonm ouonouhAuImIn no anoH nauoz mo uoowmm .HH> onnme

99
CM-Sephadex was used. Each of the salt forms of each divalent
cation was added to a reaction buffer to give final concen-

"2 M, 10"3 M and 10'“ M. The enzyme was added

trations of 10
and the solutions were incubated for 15 minutes at 30°,
before the reaction was initiated by the addition of sub-
strate.

None of the cations resulted in any significant stim-
ulation of the BAP-glycerate phoSphatase activity. Most of
the cations caused at least some inhibition at the highest
concentration except for the monovalent cations and 10"2 M
strontium. Hg++, CuI+, Pb¢+, Fe++, Fe+++, Zn.*'... and Sn?"+

'2 M and in some cases at 10‘3 M, but

were inhibitory at 10
no significance is attached to these facts, because of the
high concentrations. Cu++ and Zn‘"+ ions were inhibitory at
10'“ M. Zn++ ion was of particular interest because

P-glycolate phoSphatase had previously been found to be the
most active in the presence of Zn'H' at 10"2 and 10'3 M, as

are other phoSphatases.

Effect of Related Compounds and Other Inhibitors

The effect of a series of compounds which are either
inhibitors of other phOSphatases, structural analogs of
BAP-glyceric acid, inhibitors of the glycolate pathway,
compounds of the glycolate pathway, possible glycolate
metabolites or amino acid modifying reagents was determined
on 3-P-glycerate phOSphatase of hhgh Specific activity from
the CM-Sephadex fractionation step. An aliquot of the

100
enzyme (5 ul) in 0.10 M sodium cacodylate buffer, pH 5.9 was
treated as in the standard assay (0.75 ml final volume) with
the indicated compound (Table VIII) at three final concentra-

'2 M, 10-3 M and 10*“ M for 15 min at 30°. Then

tions of 10
substrate (5 umoles) was added to initiate the reaction. 0f
the amino acid modifiers only‘p-chloromercuribenzoate showed
inhibition (29%) at 10-2 M.

Sodium fluoride and molybdate exhibited the typical
phoSphatase inhibition but inhibition by L(+)tartrate was
less than for other phoSphatases and was only 29% at 10'2 M.
Lineweaver-Burk plots of Lt+)tartrate inhibition, as shown
in.Figure 16, indicated that it exerted a typical competitive
inhibition.

Two inhibitors of the glycolate pathway, hydroxypyri-
dinemethane sulfonate and isonicotinyl hydrazide, were inef-
fective in reducing 34P-glycerate phOSphatase activity.

None of the metabolites of glycerate or the glycolate pathway
affected the enzyme.

0f the structurally similar compounds, only arsenate,
bicarbonate, carbonate, borate, dihydroxy tartrate, and
amino-(oxy)acetic acid showed some inhibition. Isocitrate,
cis-aconitate and citrate, which stabilizes the P-glycolate
phoSphatase (114) and which are also competitive inhibitors
of P-glycolate phoSphatase, were only slightly inhibitory
at 10"2 M to BaP-glycerate phOSphatase. It seems character-
istic of BAP-glycerate phOSphatase to be stable and insensi-
tive to most of the usual inhibitors.

101
Table VIII. Effects of Other Inhibitors and Related Compounds

A 5 p1 aliquot of enzyme (S.A. = 562) in a final volume of 0. 5 m1
of 0.1 M.pH 5. 9 sodium cacodylate and 102 M. 10 3 M, and 10 4 M in
effector reagent were incubated 15 min at 50. PhOSphatase activity was
determined by adding 5 umoles 5-P-glycerate (6 7 mM final cone) and incu-
bating 10 minutes more at 500. The reaction was terminated by the addi-
tion of 0. 25 ml 10% TCA. The activities were stated as percent of con-
trol.

 

 

Concentration of Effector

 

 

 

 

 

Effector
10'2 M 10'8 M 10'4 M

Control 100 100 100
Amino Acid Modifiers

‘p-Chloromercurobenzoate 76 86 98

Iodoacetate 85 102 102

N-Ethyl maleimide 100 98 95
PhOSphatase Inhibitors

KF . 7 55 95

L(+)Tartrate 71 9h 98

Oxalate A 95 95 86

Ascorbate 100 97 95

Cysteine 98 100 100

Glutathione 99 96 95

NaMoO4, 2 28 us

Sodium arsenite 87 96 98
Inhibitors of Glycolate Pathway

Hydroxy-pyridine methane

sulfonate 100 100 98

Isonicotinyl hydrazide 91 98 100

102

Table VIII. Continued

 

Effector io'a‘M. 10'3 M 10'4 M

 

Structurally Similar or Related Compounds*

Sodium arsenate 66 9O 96
Glycidol 91 101 100
Glycerol 100 109 100
Pyruvic acid 105 100 98
B-Chloropropionate 100 97 96
L(+)Lactic acid 9h 100 95
D(-)Lactic acid 92 102 99
L-awAlanine 110 107 109
BaAlanine 115 101 109
Di-Hydroxymalic acid 100 95 92
Di-Hydroxytartaric acid Sh 65 75
meso-Tartric acid 81 99 101
(Amino)oxy-acetic acid 5h 100 100
Malonic acid 85 92 96
NaBo3 26 78 90

Products and Related MMtabolites

 

DL-Glyceric acid 97 97 97
DL-Serine 9h 95 97
Glycine 91 97 97
Glycolic acid 100 102 100
Glucose 109 102 97
Sucrose 111 105 101

Nanco8 79 89 96

103
Table VIII. Continued

 

 

Effector 10'2 M 10'3 M 10'4 M
Maleic acid 90 100 100
Malia acid 98 10A 101
ASPartate 106 110 109

Other Possible Effectors

 

Succinic acid 105 98 99
Phlorizin 89 100 100
Thioglycolate 112 107 102
DL-Hydroxybutyric acid 100 96 96
Isocitric acid 99 98 102
Citric acid 85 105 ioLI
cis-Aconitic acid 72 95 98
Citraconitic acid 96 96 102

 

*See mixed substrate assays for phOSphorylated compounds.

10b

 

 

 

 

Tartrate “J
c I
.. ‘ 40
E 20 I— ‘/
X / 20 mM
7 ‘ I
" Iiit' 'anx'550r"’
g o“ /X,o Control
1 MW.
,; I2 -
._:E?
i L I
200 400 600
it's": M“

Figure 16. Lineweaver-Burk- Plot of 1.(+) Tartrate Inhibition
The BAP-glycerate phoSphatase used had a Specific activ-

ity of 562 after CM-Sephadex fractionation. The standard assay

was used except that the reaction time was 1 minute, and

I.(+) tartrate at 10, 20, and #0 mM final concentration was

added as indicated. The phoSphate released was determined by

method B, using 100 ul aliquots of the reaction mixture.

105

Inhibition by GlycidoléP

GlycidolpP (1,2, epoxipropanedioleP*) has been reported
to be a potent inhibitor of rabbit muscle triose phOSphate
isomerase and enolase (135). When glycidoléP and P-glycerate
phoSphatase were incubated together at 23°, the enzymatic
activity was inhibited about 50% in 15 minutes and. 75% in 1
hour (Figure 17). At 9° this inactivation was much slower,
but after 5 hours the enzyme was 75% inhibited. This loss
of phOSphatase activity was not reversible by an 18 hour
dialysis of the inhibited enzyme. This slow but irreversible
inhibition probably was due to binding of the inhibitor at
or near the active site. This idea is consistent with the
kinetics of inhibition by glycidol phoSphate, which appear
to be competitive (Figure 18).

Effect of Phosphonic Acid Derivatives

Three derivatives of phoSphonic acids** were selected
on the basis of structural similarity to BAP-glycerate. The
phoSphonic acid derivatives were examined with the idea that
one of two effects might result. First, we looked for inhi-
bition of the phoSphatase since the CAP bond is supposed to
be resistant to normal phoSphatase action (136). And secondly,
we looked for an effect on the enZyme by Compound III that
would be analogous to any effect produced by the structural

 

*The sample of glycidoléP was the gracious gift of
I. A. Rose.

H
The phoSphonic acid derivatives were the gracious
gift of I. F. Isbell, Texas A & M University.

106

 

8

8

°/o of Original Activity
0:
O

N
O

 

 

 

 

Time, Hours

Figure 17. Time Dependence of Enzyme Inactivation by
Glycidcl-P

3-P-Glycerate phosphatase (3 units) was incubated
with it mM P-glycidol in 10 mM sodium cacodylate buffer at
pH 5.9 and 1 mM mm at u° and 23°. Aliquots from the
enzyme-inhibitor incubation were removed at indicated
times, assayed, and phoSphate determined by method A.

0—0 Incubated with glycidol-P at 4°

o—o Incubated with glycidol-P at 23°

A—A Incubated without glycidol-P at u°

v—w Incubated without glycidol-P at 23°

107

 

‘d(4»nflu
GMyckhM-tKDq

m
(n
l

2.nflw

N

is

l
.\.
\-

\.

  
  
   

, (umoles x mind)"l
m
C)
W

5%
l

 

J.
v

   

 

 

A// I I I I

-3oo -|00 l00 soo soo 700 900
l -l
fé’l’ M

Figure 18. Lineweaver-Burk Plots of Glycidol-P Inhibition
The enzyme (purified through the CM-Sephadex step)
was assayed in 0.3 ml reaction volumes containing the indi-
cated concentrations of 3-P-glycerate, 0.1 M sodium caco-
dylate buffer, pH 5.9. and o. 1, 2 or u mM glycidoléP. The
reaction time was 1 minute, terminated by the addition of

0.3 ml of 10% TCA. The amount of phoSphate released was
determined by method B.

108
analog aSpartate, which is a major product of the photosyn-
thetic Cu-dicarboxylic pathway in the same mesophyll cell
with the phoSphatase.

COOH COOH COOH
l l
H-CAP03H2 HzN-C4P03H2 HzN-C-H
I l I
H H H-'C-P03H2
H
I II III
PhOSphonO- Z-Amino phOSphonc- 2-Amino, 3-phoSphono-
acetic acid acetic acid propionic acid

The enzyme did not hydrolyze the phoSphonic acids to
orthophOSphate (Table IX). The phOSphonates did not inhibit
34P-glycerate phoSphatase from sugarcane leaves. However the
phOSphonates at 1 mM final concentration stimulated enzymic
hydrolysis cf BAP-glycerate, which was at a 10 mM concentra—
tion. In a more detailed experiment (Figure 19), the phos-
phonic acid stimulation became maximum as the amount of the
34P-glycerate substrate approached a saturating concentration
for the enzymatic hydrolysis. At low concentrations of
BAP-glycerate, stimulation did not occur. The significance
of this stimulation is not known. The stimulation was
reproducible but not great. Similarly aSpartate, also at
1 mM concentration, stimulated (10%) the phOSphatase (Table
VIII).

109

 

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+ :5 n.o .onoc onsocaoocn

 

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+ 26 H nonoo unnonaoonm
Amenv sum AHOHV new Ammo own cenIn so 6n
+ no on .onos onsoaacosn
o o 0 Ana Onv enno onus uncondoonm
sow com sow Annso eonIn as One nonoaoo
nonunoo nonunoo nonunoo
mo $ nns\mw1 mo & nna\mw1 «o R nn8\mw1
ouonondonaononamonmIn ououoooononaocnm ououoooononaoonm
onn84im onns4IN mousnuonsm

 

mo>nuo>nnoo once uncondoonm

 

 

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was onenno one .mommo osmnno onoonoum onu on ooooo ono3 consonaoo ononaoona one

ooouonamonm ouonoomnoImIn an anomnonohm
ouonoomnuImIn no uoonmm unone one uo>nuo>nnon ono4 uncondoonm mo unmenononm .NH onnme

110

 

.b
8
i

l

S

8

3

:‘
i!§
I“;
ll:
05‘
\l
\!
.‘a

 

pg P x mIn'”l
8
C)
1
\.°"

 

 

7

2 4 6 e 0 l5
[S], mM

{I
'1
l I I I I I _.,_I_I

Figure 19. The Michaelis Curve for 3-P-Glycerate PhOSphatase
in the Presence of PhOSphonic Acid Derivatives

The activity of 3-P-glycerate as a function of sub-
strate concentration was determined using 0.3 ml reaction
volumes containing 0.1 M sodium cacodylate buffer at pH 5.9,
2 mM of phOSphonic acid derivative and indicated concentra-
tion of substrate. The amount of phoSphate released was
determined by method B.

.0-—0 Control

A---A PhoSphonoacetate

In . nl 2-amino-phoSphonoacetate

X-a-X 2-amino ,3-phOSphonopropionate

111
Products of the Enzyme Reaction
Enzymatic hydrolysis of 3-P-g1yceric acid with the
2530-fold purified phOSphatase was complete and yielded
stoichiometric amounts of glycerate and inorganic phoSphate

under the conditions described in Table X.

Table X. Stoichiometry of 5-P-Glycerate Phosphatase

The reaction mixture contained in 2 ml: 0.52 units of 5-P-
glycerate phOSphatase, 100 umoles of Sodium cacodylate buffer at pH 5.9,
and 5-P-glycerate as indicated

 

 

 

 

 

Substrate Hydrolysis Products
5-P-G1ycerate PhOSphate (nmoles) Glycerate
(nmoles) (nmoleS)
Method A Method B Method C
1.0 0.80 0.95 1.05 1.50
2.0 1.85 1.80 2.02 2.15
5.0 5.20 2.90 5.21 5.50
u.0 h.17 5.85 5.8h u.10
6.0 6.50 5.95 6.50 6.00
8.0 7.85 8.00 7.82 7.90
10.0 10.00 9.h5 10.ho 10.10

 

In another experiment reaction mixtures of 2 ml, con-
taining 20 umoles BAP-glycerate at pH 5.9. and 1.06 units of
enzyme, were allowed to react for 0, 2, A, 6, 8, and 10
minutes. The reactions were stepped by placing the tubes in
a boiling water bath. The cooled samples were converted to

112
acid form by passage through 0.3 mm x 20 mm Dowex 50 (H+)
columns and aliquots were Spotted on Whatman No. I paper.
The chromatograms were developed and compounds located as
described in Materials and Methods. The chromatograms
showed that the hydrolysis products of BAP-glycerate
chromatographed with the Rf's of glycerate and orthophos—
phate, and that the amount of these products increased with
increasing hydrolysis time.

III. Physical Characteristics

Sucrose Density Gradient Centrifugation*

To estimate the Size of 3-P-glycerate phoSphatase,
sedimentation.ve1ocity experiments were run with sucrose
density gradients. A sample of the purest enzyme (CM-
Sephadex preparation) was layered on a 5-20% sucrose
gradient prepared in 20 mM cacodylate buffer, pH 6.3, and
1 mM EDTA. Crystalline bovine catalaee (Worthington) was
used as a marker enzyme with a known 820,,' value of 11.3 S
for a molecular weight of 250,000 (155). PhoSphatase and
catalase were placed in separate tubes (Figure 20-A) and
in the same tube (Figure 20-B). When run separately 3-P-
glycerate phOSphatase was distributed in a very broad peak
with a maximum around 13.6 S. A significant portion of the
enzyme had been pelleted to the bottom of the tube. About

 

*The author would like to thank Dr. John Boezi for
his assistance and counsel on this part of the investiga-
tion.

113

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.4 anon on .835»
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.pnoaoenw

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ooesssnoosn BenoohoInIn 0|].

ooeaeueo .drlla‘
.onnoooonn oneonepo en ooneeee cannons on» one

oopooaaoo one: mnono Ham no enonpoenm .ownnnnunoo mun Hooon confine e n« sen ooo.mm

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moannonm anonoeno_hpnonom onononm .om onnwnm

11h

 

 

 

 

 

 

 

 

 

 

 

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Fraction Number

115
81 percent of the phOSphatase that had been applied was
recovered in the gradient, and it is assumed that the rest
of the activity (19%) was centrifuged to the bottom of the
tube. When the phOSphatase and catalase were on the same
gradient. the phoSphatase profile was smeared. It appears
that something in the catalaee preparation caused some
manner of heterogeneity in the phosphatase. The catalaee
peak with the phosphatase was comparable to the catalaee
peak when run separately. and therefore no interaction of
the phosphatase with the catalaee was apparent. About 96
percent of the catalase activity was accounted for in both
gradients.

The gradient profiles of the BAP-glycerate phoSpha-
tase appeared characteristic of an aggregating system with
a multiplicity of active forms. This suggested some sort
of ionic interaction could be taking place and it was felt
that high salt concentrations might be instrumental in
overcoming the protein—protein interaction. Enzyme aliquots
of the most pure CM-Sephadex fraction were placed in a solu-
tion of 0.1 M sodium cacodylate buffer, pH 6.3, 1 mM EDEA
and 0.1 M in KCl and layered on a 5-20% sucrose gradient
prepared in 20 mM sodium cacodylate buffer. pH 6.3. 1 mM
iEDLA and 0.25 M in K01. The phosphatase and catalase were
run on separate gradients (Figure Zo-C) and they were run
together on one gradient (Figure 20-D). Greater than 90
percent of the activity of both enzymes in both.e1periments

was recovered in the peak fractions. In the presence of the

116
high salt condition. BAP-glycerate phosphatase exhibited an
320,w value of about 8.0 S. This S value is characteristic
of a molecular weight in the area of 160.000. This molecu-
lar weight is in agreement with the enzyme behavior on the
0-200 gel filtration, where it had an eluticn volume to
void volume ratio of 1.4:1 (137). However no aggregation
was observed on the Sephadex G-200 column. suggesting that
aggregation phenomenon is not Just a function of slow ionic
strength. The significance of the multiple forms, as sug-
gested by the enZyme distribution in sucrose gradients, is
not known but is of interest to note that all enzymes so
far that are involved in regulation behave in this manner

(138).

SUSAPclygcrylamide Electrophoresis

Thirtyafive microgram samples of concentrated 3-P-
glycerate phosphatase from the CM-Sephadex fraction were
denatured and reduced by incubation at 37° for 3 hours in a
solution of 1% sodium dodecylsulfate, 1% 2-mercaptoethanol
and 0.1 M potassium phOSphate buffer. pH 7.1. Samples of
the denatured and reduced enzyme and appropriate standards
treated identically were layered directly on 5% pOlyacryl-
amide gels and electrophoresed at 7.5 milliamps per gel for
3 hours and 55 min. The gels were fixed in 12% TCA and
stained in 0.125% Cccmassie brilliant blue (R-250) in 12%
TCA. After the proteins were stained for 5 hours. the gels
were destained in several changes of 12% TCA. A 550 nm scan

117
of the gel with 3AP-g1ycerate phosphatase revealed one major
and one minor band of protein (Figure 21). The movement of
the two phOSphatase bands are shown in Figure 22 relative
to the standard proteins of known molecular weight. The
major polypeptide band of the phosphatase had an estimated
molecular weight of 51.000 and the minor polypeptide band a
molecular wehght of 62,000. The relative amounts of the
major and minor bands were calculated to be 176 to ’49 by
determining the areas under the peaks of the gel scan shown
in Figure 21. The molar ratios were 4.3 to 1. It is
unknown whether the two bands are both subunits of the phos—

phatase or whether one band is an impurity.

gglyacrylamide Gel Electrophoresis

Attempts were unsuccessful to electrophores' concen-
trated BAP-glycerate phosphatase (70 mg and 105 us) from
CM—Sephadex fractionation on 5% polyacrylamide gels using
three buffer systems, B-alanine-glacial acetic acid, pH n.3,
Tris-glycine, pH 8.9 and Tris-barbitol pH 7.5. Coomassie
brilliant blue was used to stain for protein but no bands
were seen in the gels. Standard proteins were electrophoresed
simultaneously with BAP-glycerate phosphatase. These proteins
migrated and were stained by the Coomassie blue procedure.

confirming that the system.was operational.

118

 

 

 

 

 

T
o;
O .E
q ..
o’ 5
it.

 

 

 

 

Figure 21. Gel Scan of SDSAPclyacrylanide Gel Electrophoresis
of 34P~Glycerate Phosphatase

Polypeptide chains were stained with Coomassie bril-
liant blue (3.250) and the gel scanned at 550 nm.

119

 

 

 

 

quMyoekl
20
E)
9 e .0 Chain RNA Polymerase
x
E
3 l0!—
3 3—
5 Serum Albumin e‘ ,
‘5 5" 3-P-clycorato A‘\Mmr
E Phosphatase A Major
g 4__ . Chain RNA Polymerase e\
8% 3_ DNAOO. _.\
..l
22-—
l l l L l
I 2 3 4 5 6

Distance from Origin (cm)

Figure 22. Molecular Weight of Polypeptide Chains of
3-P -G lycerate P hosphatase

The semi-log plot of molecular weight of standard
proteins versus distance of migration from origin is shown.
Standards of known molecular weight were myosin 220.000
(139); B-chain RNA polymerase 16.000 (140); bovine serum
albumin 68.000 (ini): a-chain.RNm polymerase 42,000 (1&0):
and Dmase 31.000 (1h2). 3-P-Glycerate phosphatase sample
was purified through CM-Sephadex and concentrated to give
a specific activity of 7&0.

120
IV. Physiological Considerations of
QaPhosphcglzcerate Phosphatase in Sugarcane Leaves

Location in the Sugarcane Plant

Activity for BAP-glycerate phosphatase was located
almost entirely in the leaves of 6 month old. greenhouse-
grown sugarcane plants (Table XI). The tissue was
extracted by the standard method for preparing crude
extracts with a Haring Blendor and assayed immediately.
Small amounts of activity in stems and roots may be from
non-Specific phosphatases. These results are consistent
with the fact that P-glycolate phosphatase was also found
only in green leaf tissue (23, 76).

Table XI. Distribution of j-P-Glycerate Phosphatase in Sugarcane Plant

Samples of root, stem and leaf tissue (50 g) were extracted for 2
minutes at high Speed in Haring Blendor with 200 ml 20 mm sodium cacodylate
buffer, pH 6.3, l mM.EDTA, 2O mM.ascorbate and 2% Polyclar AT. The extract
was filtered through 6 layers of cheese cloth before assaying by Method A.

 

 

 

T188ue umoles PhOSphate Released x Relative
gm 1 Fresh w: x Min 1 Distribution

Leaves 11.50 1000

Stem 0.50 hh

Roots 0.15 15

 

121

Localization cf the Sugarcane 3-P-Glycerate
PhoSphatase in.Leaf Tissue

Sugarcane is an example of those plants with well
developed parenchyma sheath cells containing chloroplasts
similar to plastids in the mesophyll of a C3-p1ant. In
addition, in a sugarcane leaf, green mesophyll cells sur-
round the bundle sheath cells, and in the mesophyll cells
are located the key enzymes of the Cu-pathway of photosyn-
thesis, and chloroplasts different from those in parenchyma
sheath cells (16). In an investigation on the distribution
of enzymes between the bundle sheath.and mesophyll cells,
BAP-glycerate phosphatase was found to be predominately
located in mesophyll cells and P-glycolate phoSphatase in
the bundle sheath cells. These results as well as the dis-
tribution of other related enzymes have been.pub1ished (143)
and a reprint is attached as Appendix A. In addition to
this published work, additional experiments were done to
attempt to determine if the phosphatase was in a subcellu-
lar organelle or in the cytoplasm.

In the plants such.as sugarcane, the distribution of
BAP-glycerate phosphatase among the cell types and subcel-
lular organelles is difficult to determine, because the
grinding procedures necessary to crush these hard leaves
also break most cells and particles indiscriminately.
Methods for isolating whole, intact chloroplasts from these
types of plants are only now under development. Baldry

2312;. (125) claim to have separated heavy, starch contain-

122
ing, chloroplasts of the bundle sheath cells (CB-type of
chloroplasts) from the lighter, (non-starch containing)
chloroplasts of the mesophyll cells by density centrifuga-
tion of crude sugarcane extracts on 50% sucrose solution.
Since these chlorOplasts did not catalyze complete photo-
synthesis as 1u002 fixation in the light, retention of all
the chloroplastic enzymes is in doubt. Using their method
(Baldry'gt‘gl.. 125) sugarcane leaves, which had been exposed
to 6 hours light to ensure adequate starch formation, were
ground in a sorbitol medium and the chlorOplasts isolated
by fractional centrifugation and density centrifugation on
50% sucrose (Table XII).

Nearly all of the BAP-glycerate phosphatase and P-gly-
colate phosphatase activities were in the soluble fraction.
Further, when the chlorOplast pellet was fractionated by
density centrifugation on 50$ sucrose, only 5% cf the 3-P-
glycerate phosphatase activity was found with non-starchy
chloroplasts (interface) as compared to 18% of the P-glycolate
phosphatase. The starchy chloroplasts (mainly bundle sheath
chloroplasts) found in the density centrifugation pellet had
less than 3% of the BAP-glycerate phosphatase activity as
compared to 31% of the P-glycolate phosphatase activity.
These results exemplify the difficulties inherent in iso-
lating subcellular particles from Cu-type plants. However
the data strongly suggested that the 3-P-glycerate was a
soluble or cytoplasmic enzyme. The retention of some P-
glycolate activity by these chloroplasts is remarkable

1235

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H E

 

 

 

 

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nonmnoamsm on ooonoamnmon ones mueaaea omosH .oousnas m now w x 000: an oewnmwnuneo one

mwoes nonmnwamom nuns oeunaao mumeaaouoaao one one oeueueaeo ones onemea 058 .w x 000H

ue mouanns om now oewnmnnunoo one menu owsmwnunco ooeaw e na anemone A3\3v $Om no as on

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ooonedmnmon ones mueaaea umeaaonoHao enH .nowuewnmnnuneo cusses H .w x 000: e an oeueaaea

ones mueefiaouoHno any .uoonon wnwnez ecu n“ oeean awe: um nonooom w new Naowznfla one

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Inna mo ounce o umuwe oeume>uen ee>eoH eneunewso mo Anoem m omv mafiasen oaueUfiHaanH

mueeaaonoano
oneonewnm oeueHomH on emeueaamonm oueHoozfiwum one muonoozaoumum mo nowusonnuman .HHx enema

ll

12h
considering that this phosphatase is probably in the very
tough bundle sheath cells (Appendix A), and this supports
the conclusions tint P-glycolate phoSphatase is in or on
the CB-type chloroplasts, as observed with Spinach leaves
(Literature Review).

A second method for examining the localization of the
3aP-g1ycerate activity was by fractional centrifugation
(Table XIII). The 3éP-glycerate phosphatase was found
almost entirely in the soluble fraction and this distribu-
tion strongly supports the conclusion that the 34P-glycerate
phoSphatase is a soluble, cytoplasmic enzyme. Only 4% of
the P-glycolate phosphatase was in the chloroplast fraction
isolated by these grinding and centrifugation procedures.
This is consistent with observations that this phosphatase
is readily solubilized from the chloroplasts (76).

Similar results were obtained when sugarcane leaves
were ground in a medium used for the isolation of peroxisomes
from leaf tissue by Tolbert gt a}. (25, 26). No significant
amount of 3AP-glycerate phosphatase activity was found in
any particulate fraction (Table XIV). Only 6.3% of the 3-P-
glycerate activity was found in the fraction which contained
26% of the glycolate oxidase as a peroxisome marker. The
percentage of the cytochrome.g oxidase activity, which was
a marker enzyme for mitochondria, was 3.6 times greater
than the BAP-glycerate activity in the mitochondrial frac-
tion. Again there was no evidence to suggest that the sugar—
cane 34P-glycerate phosphatase is particulate.

125

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an oeMemoe ones memeuenamona may
w swscnnu owneuawm ones oeuenewoso; one

e nuns oeuoeuuwe mes enemwu oeueucoms one

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OOH mm m.ma OOH man oo.mmm uooooxo Hmonmnuo
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nofiuuoum
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H

 

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enmmHH meeg unmouewnm mo nowuoenuxm ownOuomH nm mo nowuewnwfiuuneu Henowuamnm .HHHx eHoeH

126

Table XIV- Distribution of j-P-Glycerate Phosphatase after Fractional
Centrifugation for Isolation of Peroxisomes

IAn 80 g sample of sugarcane leaves was ground in 200 ml 0.5 M
sucrose in 0.02 M glycylglycine, pH 7.5, for #5 seconds at high Speed in
the Haring Blendor. The extract was squeezed through 8 layers of cheese
cloth and centrifuged as described.

 

B-P-Glycerate Glycolate Cytochrome g

 

Fraction PhOSphatase Oxidase Oxidase
% of total % of total % of total

Original 100 100 100

80-100 x g (nuclei,

whole chloroplasts) 1.2 0 -

6000 x g (broken

chlorOplasts, peroxisomes) 6.3 25.7 7.8

39,000 x g

(mitochondria) 2.9 u.u Io.u

Supernatant 89.6 70.0 81.8

 

Localization of 3-P-Glycerate Phosphatase by
NonpAqueous Density Fractionation

Samples of destarched maize leaves which had been
freeze-dried and fractionated non-aqueously by the method of
Smillie (#0) were obtained from Dr. J. J. Andrews. These
dried, nonpaqueous fractions were reconstituted in 5 ml of
20 mM sodium cacodylate buffer, pH 6.3, and 1 mM ETTA and
the BAP-glycerate phosphatase and P-glycolate phOSphatase
activities determined. The results of this experiment,
along with some of the results from Dr..Andrew's investiga-

tions on these same samples are presented in Table XIV.

127
The fraction of density <1.30 is supposed to represent the
chloroplasts with some also in the 1.30 to 1.33 fraction.
The other 3 fractions are considered to be non-chloroplastic
or cytoplasmic.

The BAP-glycerate phosphatase activity does not cor-
relate with chlorophyll or chloroplast marker enzymes. It
correlates very well with the acid phosphatase distribution
reported by Slack gt 2;. who located an acid phoSphatase in
the cytoplasm (#1). If the fractions of <1.30 to 1.33 are
chloroplasts and 1.33 to >1.“0 are cytoplasmic the cytoplasm
has 89% of 3-P-glycerate phOSphatase while 87% of P-glycolate
phOSphatase is with the chloroplasts. Our investigations
(Appendix A) have shown that, in corn and sugarcane, 3-P-
glycerate phosphatase and P-glycclate phOSphatase are
present in the mescphyll and bundle sheath cells reapectively.

The P-glycclate phosphatase distribution (Table XV)
again confirmed the conclusions that the enzyme was
attached to or in the chloroplasts with the Calvin pathway.

The conclusion that 3éP-glycerate is a soluble, cyto-
plasmic enzyme was strongly supported by all four approaches
to particle isolation. This conclusion also is in agreement
with the fact that the pH cptimum is below 7.0 as are most

non-chloroplastic enzymes.

Development of the 3-P-Glycerate Phosphatase
in Etiolated Sugarcane after Illumination

Nodes of sugarcane stalks were held for 21 days in

the dark at 23° and in.vermiculite moistened with Hoagland's

1253

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om>eoq nnoo oononmuoen mo nowuenonuoenm
muflmnon one nowuonomH mnooovarnoz noome noahwnm one Hannaonoano mo nonusewnunna .>N canes

129
nutrient solution. Etiolated sugarcane "leaf" tissue grew
from these nodes, and at the time of harvest the etiolated
tissue of the poorly developed leaves was approximately
to cm in height. Tissue samples from the top of the
tissue spikes (3 g) were ground in a mortar and pestle with
washed Ottawa sand, squeezed through 6 layers of cheese
cloth and centrifuged at 14,000 x g for 10 minutes. 3-P-
Glycerate phoSphatase activity was determined over a 2 day
period during exposure to 1800 foot candles of incandescent
white light in the plant growth chamber (Figure 23). Some
P-glycerate phoSphatase activity was present in the etio-
lated tissue. This amounted to about 65 units per g fresh
weight, whereas a green leaf of the same age contained about
620 units per g fresh weight. It is not known whether this
activity of 11 or 12% of the level in normal green tissue
represents non-Specific phOSphatases or is the same 3AP-
glycerate phosphatase of the green leaf. After 2h hours
of light a small amount of chlorOphyll could be seen in the
tissue, but it was too small to be measured after extraction.
Between 12 and 2% hours of light, 3aP-glycerate phosphatase
activity about doubled. After 47 hours of exposure to
light, 34P-glycerate phosphatase activity had increased at
least h-fold, and on a gram fresh weight basis it was about
h6% of that in greenhouse tissue of the same age. The
tissue was still very light green in color. During greening.
the protein in the leaf tissue also increased in parallel
with the phosphatase activity, and after 1+7 hours of light

130

 

 

 

 

 

 

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Hours in Light After Etiolaticn

Figure 23. 34P-Glycerate Phosphatase Development from
Etiolated Sugarcane Tissue Upon Illumination

Samples (3 g) from etiolated sugarcane leaf tissue
(21 days old) from stem nodes were extracted before and
after exposure to 1800 foot candles of light. The 3—P-
glycerate phosphatase activity is expressed on the basis of
g fresh weight (0—0) and mg protein (An-A). Enzyme
activity of greenhouse grown sugarcane of same age from
same source of stem nodes was 620 units 1 g"1 fresh weight

or 63 units x mg"1 protein.

131
there was 87% as much protein as in the comparable green
leaf. It can be concluded that P-glycerate phosphatase
develops in light and, therefore, possibly functions in a
manner related to photosynthesis. This result is similar
to the light catalyzed deve10pment of P-glycclate phoSpha—
tase in etiolated wheat leaves, which does not have 3-P—
glycerate phosphatase activity (76). or the develOpment of
glycolate oxidase which is at a level of about 10% of nor-
mal in etiolated cereal leaves (75, 77).

Diurnal Variation of the Enzyme Activity

A distinct diurnal variation in 3éP-glycerate phos—
phatase activity in sugarcane leaves was found when total
activity was expressed either on a protein or chlorophyll
basis (Figure 2“). High sunlight intensity was required
for this phenomenon, and when light intensities were reduced
by heavy cloud cover, as on the second day of the experiment
shown in Figure 2“, BAP-glycerate phosphatase activity did
not increase. In these experiments the day preceding also
had at least 8 hours of strong sunlight. 3-P-Glycerate
phoSphatase activity rose in the late afternoon, reaching a
maximum in early darkness and then decreased by midnight.
The maximum increase in late afternoon was about 50% on a
protein basis and 60% on a chlorophyll basis.

Interpretations of these results will require more
physiological experiments. Careful attention was taken to

assure that the plants had sufficient water to prevent

132

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133

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134 ,
water stress, which would have occurred during middle of the
day rather than at the end of the day. The diurnal cycle
for photosynthesis would have risen in the morning, peaked
before noon, decreased around noon to 1:00 pm and peaked a
second time in early afternoon (144). P-Glycerate phospha-
tase activity began to increase when the photosynthetic rate
was decreasing and in the evening when the least photosyn-
thetic activity was expected the most phosphatase activity
was found. The results suggest that the phosphatase may not
be directly associated with 002 fixation, but possibly is
involved in regulating starch formation (or breakdown). It
is late in the day that starch formation may be occurring in
mesophyll chloroplasts (62). Perhaps the phosphatase is an
enzymatic mechanism for reducing the rate of 002 fixation or
making it more efficient (no carbon through glycolate).
There may also be a need based on permeability for the glycer-
ate to be in a non-phosphorylated form. Certainly the
hydrolysis of 34P-glycerate, the first product of photosyn-
thesis would drastically curtail the 03-photosynthetic car-
bon cycle. However neither of these hypotheses is supported
by the fact that the phosphatase is located in the mesophyll
cells and the C3-carbcn cycle and the starch containing
chloroplasts are located in the bundle sheath cells.

The diurnal variation in BAP-glycerate phosphatase
activity occurs in spite of the excellent stability of this
enzyme in homogenates or when partially purified. These
facts suggest that the regulation of the phosphatase activity

135
{22;1312 is probably by some effector rather than by protein
turnover; but protein turnover cannot be ruled out with the
present information.

Because of the diurnal variation in 3-P-glycerate
phoSphatase activity, plants, whenever possible, were har-
vested in the afternoon for enzyme preparations. My major
professor was not sure that this was a valid excuse for not
coming to work until noon, but he was unavailable for comment

after midnight.

PART B:
THE DISTRIBUTION OF 3-P-GLKCERATE.AND
P-GLICOLATE‘PHOSPHATASE IN VARIOUS PLANTS

In the Literature Review of this thesis is cited some
of the evidence for similarities between the glycolate path-
way, peroxisomes and photorespiraticn. Investigations to
establish the extent of this correlation are underway in
numerous laboratories. One of the approaches initiated by
us in 1966 was to determine the relative amounts of P-glyco-
late phosphatase and 3aP-glycerate phoSphatase activities
in different leaf tissues. Since P-glycolate seems to be
the precursor of glycolate (22), then.P-glycolate phosphatase
should be present in those cells or C3-plants with the glyco-
late pathway, peroxisomes and cog-photorespiraticn. Likewise
non-COg-photoreSpiring Cu-plants or cells would be expected
to have less‘P-glycolate phosphatase and perhaps more 3-P-
glycerate phosphatase, if it were substituting for the other
phOSphatase. This hypothesis was supported by our initial
investigations with sugarcane (145). Sugarcane leaves had
low levels of peroxisomal enzymes (Appendix A) and also rela-
tive low levels of P-glycolate phosphatase (Table XV). How-
ever we did find in sugarcane leaves a large amount of phos-
phatase activity towards 34P-glycerate. These findings
prompted the isolation, partial purification and character—

ization of 3AP-glycerate phosphatase from sugarcane as pre-

sented in.Part A.

136

137

A survey of plants for the two phosphatases was based
upon the hypothesis that other COz-photorespiring plants
should have hhgh levels of P-glycolate phosphatase activity
relative to the levels of BAP-glycerate phosphatase. Non-
COz-photorespiring plants it was postulated would have high
levels of BAP-glycerate phosphatase and low levels of the
P-glycclate phOSphatase activity.* .Another purpose of the
survey was to determine the feasibility of using the rela-
tive levels of the two phOSphatases as one criterion for
distinguishing between C02-photorespiring and non-COZ-photo-
reapiring plants. We have been intrigued by the fact that
glycerate is a major product of the glycolate pathway, but
it can also be formed from enzymatic hydrolysis of 34P-
glycerate, a major photosynthesis product, essentially by-
passing the glycolate pathway (Figure 2).

I. Special Materials and Methods

The plants used for the survey were all obtained
locally in the greenhouse or from field plots. They were
harvested during the middle part of the day and assayed
within an hour of harvest. The washed, deribbed leaf tis-
sue was diced into 2 x 2 cm pieces and homogenized at high

speed in a Waring Blendor for 2 minutes in 4 to 5 volumes of

 

*Initially the survey was aided by the diligent efforts
of Dennis Gremel, an Honors College student at Michigan State
University, who is now in graduate school in Biochemistry.

138
grinding medium at 4°. The grinding medium was 20 mM sodium
cacodylate buffer, pH 6.3, and 1 mM ETTA. For some plants
(where noted) 2% Polyclar AT and 20 mM ascorbate were added
to protect against the oxidation of the phenolic compounds.
The homogenate was filtered through 6 layers of cheese cloth
and the phOSphatase activities determined in the crude plant
extract by Method A. For data in Experiment II, Table XVI,
the residue remaining in the cheese cloth after the Waring
Blendor homogenization was re-extracted by putting it
repeatedly through a roller mill (described in Appendix A)
while washing the residue as it was squeezed on the rollers
until the chlorophyll was visually all extracted from the
tissue. .All enzyme assays were at least duplicated and
most plants were examined using triplicated samples and

extractions.
II. Results and Discussion

During part of the survey for the two phoSphatases
(Experiment I, Table XVI) only the extract from Waring
Blendor homogenate of whole leaves were assayed. Later,
from our own work (Appendix A) and from ngrkman's (58) .
it was realized that in the Cu-plant the bundle sheath
cells required vigorous grinding by mortar and pestle or
roller mill for complete breakage. A differential grinding
procedure was developed (Appendix A) employing first the
Waring Blendor to break mainly mesophyll cells and then a
roller mill extraction to break the remaining bundle sheath

139

 

 

 

 

 

 

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Table XVI. Continued

ilhi

 

Waring Blendor Extraction -- Experiment I

 

 

 

Plants B-PGA P-glyc 5-PGA/P-glyc
S.A. S.A. Ratio
Aquatic Plants
Elodea 25231 #6 1h2 1/5.1
Sagittaria 1h 46 1/5.5
Algae
ahidmxdomonas reinhardtii 1h 11h 1/8.1
Ankistrodeemus braunii 15 54 1/2.2
Chlorella Ezrenoidosa 7 117 1/16.7
Scenedeamus 12 207 1/17.2
Crassulacean Plants
Kalanchoe vulcanb 15 26 l/l.7
Br 0 llump 5 9 1/1.8
Sedum apectabileb 98 180 1/1.8
Liverwort
Merchantiab 126 he 5/1
Trees
White oakb 15 150 1/10
Red III-pie" 5 23 1/h.6
Mountain ashb 240 187 1.5/1
Cottonwoodb 11 176 1/16
American slap 52 87 1/2.7
‘ aed spruceb 9 31 1/3.h

 

b20 In.aacorbate and 2$ (w/v) polyclar AT added to grinding medium.

142
cells (Experiment II. Table XVI).

The phosphatase specific activity is reported on a
per mg chlorophyll basis, since we felt the plant's photo-
synthetic ability is more closely related to its chloro-
phyll content than protein. The data was summarized by
grouping the plants in.Table XVI according to their ability
to photorespire or by their general type. The data is also
summarized by the relative ratio of 3éP-glycerate phospha-
tase activity to P-glycolate phosphatase. In general C3—
plants known to have 002-photore8piration had very high
P-glycolate phoSphatase activities, 3 to 23 umoles phos-
phate released per mg chlorophyll per min. Rates of 002
fixation probably and photcphosphorylation rates range
between 2 to 6 umoles per mg chlorophyll per min. Thus in
general the P-glycolate phosphatase activity considerably
exceeds maximum photosynthetic rates. In these C3-p1ants
BéP-glycerate phoSphatase activity was 1/2 to 1/8 as much
as the P-glycclate phOSphatase. In a group of known C4“
plants without 002-photorespiration there was more 3-P-
glycerate phosphatase and much less P-glycolate phosphatase,
so that the 34P-glycerate:P-glycolate phosphatase ratios in
these plants were nearer 2/1 to 4/1. This approximate cor-
relation was valid for the two well-studiedIAtriplex
species (58). For;A. patula with Goa-photorespiraticn the
3éP-glycerate:P-glycolate ratio was 1/1.2 while for'A, £2522
without cog-photorespiraticn the ratio was 2.9/1. However
the phoSphatase activities in the 03-bundle sheath cells of

143
A. £2232 had a ratio of 1/2 favoring‘P-glycolate phoSphatase
as predicted by the peroxisomal activity in these CB-cells
within this (tn-plant.

Spinach and sunflower are good sources of stable
peroxisomes (26) and contain high levels of P-glycolate
phoSphatase in comparison to the BAP-glycerate phoSphatase.
Likewise wheat, tobacco and soybean contain high levels of
glycolate pathway enzymes and high Specific activity of P-
glycolate phoSphatase. In all of these CB-plants the Waring
Blendor adequately broke all the cells (EXperiment II, in
Table XVI) and no further release of enzyme occurred with
the roller mill. In contrast, sugarcane, sorghum, sudan
grass and pigweed which are low in peroxisomes and glycolate
pathway enZymes (26), had low levels of P-glycolate phospha-
tase and relatively high levels of 3eP-glycerate phosphatase.
In addition it was necessary to use the roller mill to break
the bundle sheath cells to release much of the P-glycolate
phosphatase activity.

From leaves of 15 soybean.varieties, C3-plants, the
P-glycerate phosphatase to P-glycolate phosphatase ratio
also varied as predicted from 1:2 to 1:5 (Figure 26). Bush
beans (Phaseolus vulgaris, v. Sanalac) are characterized by
having large amounts of both phosphatases. Beans appear to
be an exception (Table XVI) to this hypothesis. In this
bean leaf both phoSphatases were equally as active, and
BAP-glycerate phosphatase activity was almost double the
activity in sugarcane. The P-glycolate phosphatase was

144

almost double the activity in sugarcane. The P-glycolate
phosphatase was partially inactivated by the routine long
extraction time (Figure 25-A) which.was employed to solu-
bilize the phoSphatase from corn or cane (Figure 25-C).
After 30 seconds of grinding of Sanilac bean leaves the
amount of both enzymes was about equal. and in Experiment
II (Table XVI) both phOSphatases had the same activity after
2 minutes. P-Glycolate phosphatase from tobacco leaves
undergoes similar inactivation, but not to such a degree
that the ratio is greatly affected. The extraction time
versus percent activity plots (Figure 25) were done on a
large portion of the plants examined, especially those that
did not seem to follow the predicted pattern. Figure 25-B
and C, were typical of the plots for other plants examined.

In extracts of bundle sheath cells of Cn-plants
obtained by the roller mill, there was more P-glycolate
phosphatase activity than 3aP-glycerate phosphatase. This
distribution of the phosphatases is in complete agreement
with the concept that enzymes for the glycolate pathway
and the C3-photosynthetic carbon cycle are located in the
bundle sheath cells and the Cg-photosynthetic carbon cycle
in the mesophyll cells (Table 1). It is from such results
that it is postulated that plants which utilize the Cn-acid
pathway will have higher levels of BAP-glycerate phosphatase.
Plants or cells utilizing the 03- or Calvin cycle pathway
will have higher levels of P-glycolate phosphatase activity.

Some plants have not been characterized by the plant

145

Figure 25. Extraction Time Versus Percent of the'Phosphatase
Activity Solubilized for 3 Types of Plants*

A. Bean (phaseolus vulgaris, V. Sanalac) showing inac-
tivation of P-glycolate phosphatase (A—A) after 30 seconds
of homogenization and 3eP-glycerate phosphatase (e__.e) which
was stable.

B. Typical extraction time versus percent activity
solubilized from leaves of C3-type plants (e.g.. spinach),
showing no inactivation of the phOSphatase.

C. Typical extraction time versus percent activity
solubilized from leaves of a Cu-plant (9.8.. corn), showing

no inactivation of enzyme but incomplete extraction.**

 

tAll points are obtained by averaging duplicate assays
of triplicated samples.

**Figure 7-B shows results of roller mill extraction
after incomplete Waring Blendor extraction.

IOO /3K:I:_‘
so . \‘
S-P-Glycercte /
6° P-Olycolate
4o ..
ZO-A. Bean
>. 1 l l l __
.1:
>
I: IOO - ____.
80- e
./ ‘
:5 6° F. e 3-P-6cherate
8 40 a P-thcclcte
~g 2° 8. Spinach
. l L I l
0
°\ " e
'°° 0. Corn ,/
80

60
4O
20

146

 

 

 

 

.. o '
_ fl-Gucerate
O

'- h

A

 

/
:/‘ \ P-Glycclate
I I l I
30 60 90 I20

Seconds

147
physiologists according to their manifestation of cog-photo-
respiraticn. Some of these plants are in a third group in
Table XVI. According to their phoSphatase activities
Bermuda grass and Bent grass should not have Cog-photores-
piraticn. Indeed they are in sub-familiar of Chlorideae and
Agrostideae of which there are many examples of the C4-
pathway (57). On the other hand Poa and Merion bluegrasses
with high P-glycolate phosphatase are of sub-families of the
Festuceae which is generally considered to be comprised of
C3-pathway plants (57). The two aquatic plants examined
had phosphatase activities of typical C3-pathway plants.

The unicellular algae examined all actively biosyn-
thesize glycolate and have high levels of P-glycolate phos-
phatase. These algae are considered non—photoreSpiring
however, since they either excrete glycolate or metabolize
it slowly by a glycolate dehydrogenase (146). The primitive,
non-vascular liverwort (Marchantia) however had higher
levels of 3eP-g1ycerate phosphatase. Other investigators in
Tolbert's laboratory also find low levels of glycolate
oxidase (no glycolate dehydrogenase) in this tissue (8. Wardell
and N. E. Tolbert, unpublished). The crassulacean plants
with their B-carboxylation system for acid accumulation
might be thought of as Cu-plants, yet they appear to be
C3-pathway plants according to their relative phOSphatase
activities.

Among the Cu-plants without photorespiration, corn

leaves was an exception to the above hypothesis. It had

148
relatively low levels of BAP-glycerate phosphatase activity,
and even the amount of extractable P—glycolate phosphatase
was low. However the BAP-glycerate phosphatase was located
primarily in the Cu-mesophyll cells and the P-glycolate
phOSphatase in the C3-bund1e sheath cells (Appendix A).
This distribution is consistent with the hypothesis. The
reason for the lower levels of total 3-P-g1ycerate phoSpha-
tase activity is not understood.

Leaves of trees are grouped separately. The 002
compensation point of these leaves is as high as 150 ppm.
and. as such. tree leaves (North.American. temperate zone
trees) are in general the most actively photorespiring leaf
tissue known. As predicted, they have high levels of P-
glycolate phosphatase and low levels of P-glycerate phos-
phatase. These values must be qualified by the difficulty
in obtaining adequate amounts of active enzymes from
homogenate of tree leaves. The tree leaves were ground. in
the presence of Polyclar AT in order to reduce the tanning
action. These homogenates were still so full of tannic
material that all particles and most of the protein were
coagulated and enzyme assays could only be run on the small
portion of the protein which is not precipitated. Because
of this problem with tree leaves many enzymes, including the
peroxisomal enzymes, cannot be detected in tree leaf extracts
(Tolbert. unpublished). Likewise unknown and different
amounts of the phosphatases may have been precipitated by
the tannins and the levels reported in Table XVI can only

M9
be considered as exploratory values.

The fact that high levels of P-glycolate phosphatase
was present in plants which have high peroxisomal reapira-
tion (oxidation of glycolate in the light = photoreSpira-
tion) supports the conclusion that P-glycolate is the
immediate precursor of glycolate. The fact also that 3-P-
glycerate phosphatase was present at relatively high
levels (except corn) whenever the Cu-dicarboxylic acid path_
way is present supports the conclusion that its presence is

related to the Cu-pathway.

III. Relative Levels of 3aP-Glycerate and
P-Glycolate Phosphatases as a Function
of the Rate of Photosynthesis in Soybeans

The initial results of the survey of plants for the
relative levels of BAP-glycerate and P-glycolate phospha-
tases activities supported the conclusion that 3aP-glycerate
phosphatase was the more active in Cu-plants (e.g.. sugar-
cane. sorghum) which are considered more efficient or have
a lower 002 compensation point than 03-p1ants (Literature
Review). In an attempt to apply this hypothesis it was
postulated that the relative levels of the two phoSphatases
might be a criterion of photosynthetic efficiency or growth.
Professor R. H. Hageman suggested to us that varieties of
soybeans which had a range of known €02 fixation.rates (1&7),
would fit the requirements for such an experiment. Through

the cooperation of Dr. R. H. Hageman of the Agronomy

150
Department and w. L. Ogren of the U.S.D.A. Regional Soybean
Research Laboratory at the University of Illinois, Urbana.
the activities of BAP-glycerate phosphatase and P-glycolate
phoSphatase in 15 varieties of soybeans (Glycine Egg.
L. Merrill) were determined. The soybean varieties had
rates of photosynthesis from in to 2# umoles 002 fixed per
minute per square decimeter of leaf tissue (1h7). All of
the varieties were planted at the same time in the same
field plots. Three. 20 g leaf (7-9 leaves) samples of each
of the varieties and 2 complete replications were used for
each experiment. The activities of the phoSphatases were
determined in June on small plants (5-10 cm in height) and
in July on large plants. Just before the onset of flowering.
The results of the July experiment are presented in Figure
26 in which the phosphatase activities are expressed on a
per mg protein or chlorophyll basis. Although there is
considerable scatter in the points. a regression analysis
(Table XVII) of the data showed that they were significant
at the 0.05 level.

Soybean varieties with increasing rates of 002 fixa-
tion had higher levels of P-glycolate phosphatase and lower
levels of BAP-glycerate phosphatase activities. The initial
hypothesis was that plants with higher rates of 002 fixation
would have relatively more 34P-g1ycerate phosphatase as do
sugarcane and sorghum.while those plants with low C02 fixa-
tion rates would have more'P-glycolate phosphatase. because
considerable amounts of the 002 fixed would be respired by

151

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153

Table XVII. Correlation of j-P-Glycerate PhOSphatase and P-Glycolate
Phosphatase Activities with Photosynthesis in 15 Soybean

Varieties
_____.——-——-—'—_ =—‘

3-P-G1ycerate PhOSphatase

 

June July

P-Glycolate Phosphatase

 

 

 

I II I II

June July

 

I II I II

 

Prot. s.A. -.755** -.625* -.u85 -.363

Chl. S.A. -.681** -.522* -.h13 -.57o

 

June AVg. July Avg.
PrOt. SOAO '0702** -0hh1
Chl. S.A. -.660** -.399

Overall Avg.

 

Prote 80A. '0580*

Chl. Se A. -0512

.5A6 .20} .506* .572*
.585 .19h .558* .h15

June Avg. July Avg.
.285 .596*
-296 .ugu

Overall AVg.

 

.552*
.h77

 

+Replications I and II were run with 3 samples each time in both June

and July.
*Significant at .05 level (.51h).

**Significant at .01 level (.6h1).

15a
the flow of carbon through the glycolate pathway and peroxi-
somal reSpiration. The results were Just the opposite.

There are some qualifications to the results which
could have a considerable influence on the validity of the
relationship indicated. First of all. the data of Curtis
22 gl. (1“?) for the photosynthesis rates were considered
to be absolute. However investigators at the University of
Minnesota and Iowa State disagree with the rates of some of
the varieties (R. H. Hageman. personal communication).
Secondly. the rates that Curtis reported were from seedlings
grown in a growth chamber whereas field grown plants were
used for the phoSphatase assays. Third, the genotypes used
are quite variable, with regard to maturity types, growth
requirements and grain composition. Certainly the metabol-
ism must also vary with genotype. Considering these factors
it is amazing that any relationship or correlation was found
at all.

The initial hypothesis for this experiment had
assumed that the higher the net rate of 002 fixation the
more efficient would be the net photosynthetic growth and
peroxisomal or glycolate metabolism would be less. The
assumption was based upon the fact that glycerate. glycine.
serine and Cl-moieties are all products of the glycolate
pathway which starts with the hydrolysis of two‘P-glycolate
(Figure 2). During this conversion of two P-glycolates
molecules to one molecule of glycerate. considerable

energy is lost and one 002 is respired. To make glycerate

155
by the hydrolysis of one BAP-glycerate molecule is much more
efficient, both from the standpoint of a carbon conservation
and of phoSphorylation. From the data in Figure 26 this
assumption appears to be in error. The original hypothesis
would have been reversed to accomodate this data to state
that soybean.plants with.more P-glycolate phoSphatase activ-
ity and presumably peroxisomal respiration are photosynthe-
tically more active. If peroxisomal metabolism is essential
for photosynthesis and polysaccharide synthesis, then
increasing capacity for glycolate metabolism should coincide
with increase rate of photosynthesis.

PART C:
A PARTICULATE 3éP-GLYCERATE PHOSPHATASE
FROM SPINACH LEAVES

During my thesis research. isolation and characteri-
zation of leaf peroxisomes has been in progress by others
in the laboratory (25, 26, 82). The metabolic sequence of
the glycolate pathway associated with the peroxisomes
begins with glycolate and ends with glycerate (Figure 2).
Consequently the cellular location of P-glycolate and 3-P-
glycerate phOSphatase activities have been of particular
concern to the peroxisomal investigations. Neither phos-
phatase was found in the peroxisomes, but rather P-glycolate
phOSphatase is located in the chloroplast and part of the
BAP-glycerate phosphatase in an apparent starch grain from

spinach leaf extracts.

wcohte PhoSphatase in Chloroplasts

P-Glycolate phosphatase was first reported to be
associated with the chloroplasts of spinach by In 33 a}.
(76). Thompson and Whittingham (70) confirmed by non-
aqueous density gradient techniques the location of the
P-glycolate phosphatase with the chloroplasts (89-97%).
Using an aqueous sorbitol medium we also concluded that
the enzyme is with chloroplasts (Table XVIII) but that the
BAP-glycerate phosphatase did not appear to be with the
chloroplasts. The recovery of only 12% of the P-glycolate
phosphatase in the chloroplast fraction indicated that the

156

157'
enzyme was loosely associated with the plastids. However
the advantage of sorbitol medium over NaCl or sucrose iso-
lation medium was that most of the P-glycolate phOSphatase
activity with the chloroplast fraction remained with the
chloroplasts upon repeated washings which completely

removed other glycolate metabolizing enzymes.

Table XVIII. PhOSphatase Activity of Isolated Spinach Chloroplasts Pre-
pared in Sorbitol Medium as Described in Materials and

 

 

 

 

Methods
I I:
P-Glycolate E-P-Glycerate
PhOSphatase PhOSphatase
Fraction
Units % Units %
Original extraction 11,350 100 2,h80 100
Supernatant 8,580 77 1,080 A;
Chloroplasts 1,h00 12 (100)* #5 2
1x Washed
chloroPlasts 1,500 9.1 (7h) Trace
2x washed
chlor0p1asts 950 8.h (68) Trace
5x Washed ‘
chloroplasts 916** 8.1 (66) Trace

 

*Values in parenthesis are percent of enzyme in chloroplast fraction
after the first centrifugation.

**Equiva1ent to 39.6 umoles of phOSphate released x minute-l x mg-l
chlorophyll.

158

3-P-Glycerate Phosphatase in Starch Pellet

During the isolation and characterization of the
spinach leaf peroxisomes from the 6000 x g fraction by
Tolbert gt_al. (25), the various fractions from the iso-
pycnic sucrose density gradients were assayed to determine
the activities of the 3-P-glycerate and P-glycolate phos-
phatases. Neither phoSphatase was found in the peroxi-
somal. mitochondrial. or chloroplast fractions under these
conditions. However as much as 38% of the 3-P-glycerate
phosphatase activity was in the 6000 x g pellet (Table
XIX). About half of this activity was apparently soluble
enzyme and remained on the top of the discontinuous sucrose
gradient. The other half of the activity pelletted to the
bottom of the sucrose gradient through 2.3 to 2.5 M sucrose.
Microscopic examination of the small pellet indicated that
the major constituent was small starch-like grains, although
the pellet was somewhat gray in contrast to the usual white
expected from starch. The fact that it gave a blue-purple
stain with iodine and KI agrees with the idea that the only
logical particle which could pass through such high.density
sucrose would be starch or a similar polysaccharide. Thus
the particle has been termed the starch particle and the
pellet, the starch pellet. The few whole cells. cell
fragments and broken chloroplasts that were present in the
pellet should not have been able to sediment through the
2.5 M sucrose and probably moved down the side of the

centrifuge tube. The nature and composition of the starch

159
pellet and the BAP-glycerate phoSphatase with it were sub-
jected to further investigation because the phOSphatase

activity in the pellet appeared to be a new discovery.

Table XIX. Distribution of j-P-Glycerate PhOSphatase in Peroxisome
Preparation

The peroxisomes were prepared by the method of Tolbert SE 11. (25)
from 500 grams of Longstanding Bloomsdale Spinach. The gradient pellet
was resuSpended in 0.8 M sucrose containing 20 mM sodium cacodylate
buffer, pH 6.5, and 1 mM EDTA.

 

 

E-P-Glycerate PhosPhatase

 

 

Fraction

Units % of Total % of 6000 x g S.A.

Pellet

Original 7,280 100 -- 0.027
6000 x g fraction 2,850 58 100 0.265
All gradient fractions Trace -- -- --
Gradient supernatant 1,200 18.5 #2 --
Gradient pellet 1,370 18.h hB 1.280

 

The stability and storage characteristics of the 3-P-
glycerate phoSphatase of the starch pellet were very good.
The pellets from the preparation of peroxisomes on sucrose
gradients have been stored frozen for as long as 3 months
before resuspending without loss of BAP-glycerate phoSphatase
activity. The resuspended particulate enzyme was stable at
0° until microbial growth started (several weeks) and the

frozen suspensions were stable indefinitely. Preparations

160
of the solubilized phosphatase or partially purified enzyme
from the starch particles were stable and could be stored

frozen at -18°.

pH Optimum
The pH activity curve for the 3-P-glycerate phoSpha-

tase in the Spinach starch particles had a sharp optimum
around pH 5.8 (Figure 27). The addition of Mgi+ did not
effect the optimum or the activity in any way. The pH
optimum is very similar to that of the soluble sugarcane
BAP-glycerate phosphatase (pH 5.9). The enzyme was stable
at 4° at pH 6.3 or 7.0.

Kinetics of Enzyme

The particulate BAP-glycerate phoSphatase activity
was a linear function of enzyme concentration.(Figure 28).
The particulate enzyme. the dialyzed particulate enzyme and
acetone precipitated enzyme from the particles all showed a
linear function with enzyme concentration. Normal enzyme
saturation kinetics were obtained with the particulate
enzyme (Figure 29qA) and the apparent Km for B-P-glycerate
was 9 x 10-4 M (Figure 29-3).

Effect of Divalent Cations

The 3-P-glycerate phosphatase in the starch particle
did not show any requirement for a divalent cation (Table
XX). The addition of 1 mM EDTA or dialysis against 20
volumes of 1 mM EDTA did not inhibit the enzyme activity,

161

Figure 27. The pH Activity Curve for 3-P-Glycerate Phospha-
tase in Starch Particles

The enzyme activity was assayed in the particulate
form and in 3 buffer systems: 0.1 M sodium cacodylate +
0.1 M sodium acetate; 0.2 M sodium cacodylate: 0.1 M sodium
cacodylate + 0.1 M glycylglycine. The activities in the

different buffers were averaged for the overlapping points.

Figure 28. Linearity of the BAP-Glycerate Phosphatase Assay
Increasing amounts of the enzyme in.3 forms were

assayed. The particulate enzyme (0———O) was suSpended in
0.8 M sucrose. 1 mM EDTA and 20 mM sodium cacodylate buffer
at pH 6.3. The particulate enzyme was dialyzed against 20
volumes of 20 mM sodium cacodylate and 1 mM EDTA for 20 hours
and then assayed.(a———e). The acetone fractionated enzyme
(I-——I) had been solubilized by sonification of the starch

particles and precipitated by acetone as described in the
text.

Relative Activity

umles Phosphate x min"

 

a

 

 

 

 

 

 

fl
30- l.
60— ‘. \
l
40— \
.
20~— \
1 1 1 ~o 1
4 6 8 l0
pH
"6_ Acetone I
Fmtion7/
l.2-— I ’

 

 

......l /‘
/./

Particulate

l l J
20 30 40
pl of Enzyme

 

 

163

Figure 29. Kinetic Plots for the Spinach Particulate
3-PuG1ycerateIPhosphatase

A. The initial velocity as a funetion of substrate
concentration was measured in 0.3 ml reaction mixtures cone
taining 0.1 ml substrate of indicated concentration. 0.15 ml
0.2 M sodium cacodylate. pH 5.9 and 0.05 ml of suspended
enzyme. The reaction.was terminated after 1 minute with
0.3 ml.10$ TCA. The phosphate was determined by method B.

B. A Lineweaver-Burke plot of initial velocity versus
substrate concentration. The apparent Km is about 9 x 10’“ M

for the particulate enzyme.

161i

 

 

 

 

 

 

E A /.r ‘e
x 6 /.’.
e
i F V"
_ 4»—
a? I
°' 2
3
x 1 L L A l l
‘B 2 4 6 8 l0
- [8], mM
9 32.. B
{E /
E
x 24- ! ./e
U)
.93
g '61.. ../
a l"
8 Km=9x|O-4_M_
l-.
.5
1

 

 

 

l 1 l 1
0.4 0.8 I .2 LG 2.0
I/[s]. .m_M_"

1165

Table XX. The Effect of Divalent Cations on the B-P-Glycerate Ph03phatase
in Starch Particles

Aliquots of the particles which had been dialyzed against 1 mM
EDTA were equilibrated at 500 for 15 minutes in 0.5 m1 of a solution con-
taining 0.15 M sodium cacodylate buffer, pH 5.9, with 5 mM or 0.5 mM
cation. The reaction was initiated b the addition of 0.25 ml substrate
(10 umoles), ran for 10 minutes at 50 and was terminated by the addition
of 0.25 ml of 10% TCA.

 

 

Relative Activity

 

 

Addition
5 x 10-3 M.Cation 5 x 10'4 M Cation

Control 100 100
Control + EDTA 102 100
2nso4 5 15
Coso4 78 85
CuSO4 1h 15
Pb(Acetate)2 10h 99
CaClg 98 101
MnClg 102 101
Mgso4 10h 101

Niso4 80 101

 

166
but. in fact, increased the activity about 10%. As observed
for the soluble sugarcane BAP-glycerate phoSphatase, Zn++
was inhibitory at 1 mM and Cu?"+ also caused considerable

inhibition. greater than 50% even at 0.5 mM.

Substrate Specificity

The relative Specificity of the starch particle 3-P-
glycerate phoSphatase (Table XXI) was similar to that for
the soluble phoSphatase from sugarcane leaves. The sub-
strates were also assayed in the presence of 1 mM MgSOg.
with no effect on the hydrolysis of BAP-glycerate and mixed
effects on the other substrates. The partially purified
particulate enzyme was not Specific for 3-P-glycerate, but
it is probably the preferred physiological substrate. Since
there was a possibility that the nuclei of the cells may
have been part of the particles. the nucleotidase and dies—
terase activities were investigated. but no significant
activity was found by the assays used. However. the par-
ticulate enzyme may not be completely free of other phoSpha-
tase activity.

Solubilizing the Particulate QaP-Glyoerate Phosphatase

In order to adequately resuSpend the starch particles,
it was necessary to use approximately 0.8 M sucrose and
buffer. “When the particles were suSpended in buffer alone
and sonicated for 20 minutes. only 50% of the BAP-glycerate
phoSphatase was solubilized to the extent that it was not
pelleted by a 10.000 x g. 20 minute centrifugation. However

167

Table XXI. Substrate Specificity of the Starch Particle 5-P-Glycerate
Phosphatase

en umoles (15.5 mM) of all substrates were used in the assays.
The Mg was equilibrated 15 minutes with the enzyme before the reac-
tion was initiated with Substrate. The enzyme was used in the resus-
pended particulate form.

 

 

Relative Activity

 

 

Substrate - Mg++ +~Mg++
5-P-Glycerate (control) 100 100
P-Glycolate h 5
2-P-Clycerate 1h 15
G1ucose-6-ph03phate A7 to
Fructose-1,6-diph08phate 67 66
B-Glycerol phOSphate 50 56
‘B-NitrophenylphOSphate 96 112
bisjp-NitrOphenylphOSPhate 9 8
Adenosine triphosphate 70 52
Adenosine diphOSphate 67 62
5'2Adenylic acid 60 59
5'-Deoxyadeny1ic acid A h
5'-Adenylic acid 50 52

3"Deoxyadenylic acid 3 8

 

168
if the starch pellet was suSpended in 0.8 M to 1.3 M sucrose.
90 to 94% of the phoSphatase was solubilized when sonicated.
Homogenization in a glass Potter Elvejehm homogenizer did
not release the enzyme from the particles. nor did repeated
passage through a French pressure cell. Repeated freezing
and thawing did not solubilize the enzyme. Dialysis of the
suSpended particles against 20 volumes of 20 mM sodium
cacodylate buffer. pH 6.3. and 1 mM EDTA did not release the
enzyme but did result in about a 10% increase in the activ-
ity; These procedures succeeded in breaking all whole cells
and chloroplasts in the suSpension. Limited solubilization
of the phoSphatase from the starch grains seemed to be
favored at pH values above 7 (Figure 30). .At pH 7.0 to 7.5
a maximum of 36% of the enzyme was released from the particles.
Above pH 7.5 the stability of the particulate phoSphatase
began to decrease with a parallel decrease in the activity
that was solubilized.

Incubation of the starch particle suSpension with
B-amylase at 4° in sodium acetate buffer at pH 5.5 resulted
in solubilization of the BAP-glycerate phoSphatase and the
concomitant release of increasing amounts of reducing sugar
(Figure 31). These results tend to support the idea that
the particle contains some form of starch or similar poly-
saccharide. The reason for loss of activity at 48 hours is
not known.

The effect of salt concentration on the solubiliza-

tion.of the particulate (starch grain) BAP-glycerate phos-

169

Figure 30. Solubilization of the Starch Grain 3-P-Glycerate
PhoSphatase as a Function of pH

The resuSpended particulate enzyme was diluted 5 fold
into 0.2 M buffers (cacodylate, acetate or glycylglycine) of
the indicated pH and incubated for 1 hour at 4°. The particle
suSpension (0—0) and the supernatnat (0---0) of 10.000 x g

for 20 minutes centrifugation of the suSpension were assayed.

Figure 31. Solubilization of the Starch Grain 3-P-Glycerate
Phosphatase by B-Amylase

Samples (0.5 ml) of the particulate 3-P-glycerate phos-
phatase were incubated in 50 mM acetate buffer. pH 5.5. with
700 units of B-amylase (Worthington) at 4° for a 48 hour period.
The phoSphatase activity of theresuSpended particles (0—0)
and the supernatant (h—‘) after 10.000 x g for 20 minutes
centrifugation of the suSpension were assayed. Reducing sugar

(...-I) was determined by Nelson's test (120).

pg P Released x min"I

pg P Released x min"

8

(D
0

§

400

§

170

 

_. Suspension

./ .~.~

.- Solubilized
E

 

4.5 . 5.5 6.5 75 8.5
pH

 

l" I

530/ \ Solubilized

 

 

 

 

0 I2 24 36 48
Hours

pg Reducing Sugars

171
phatase is not completely established (Figure 32). Incuba-
tion of the starch particles in 0.25 M MgClz solubilized
100% of the phoSphatase (Curve II. Figure 32-B). However
as the MgC12 concentration was increased to 0.35 M MgC12 or
greater, the total soluble activity (supernatant) increased
15 to 20% over the total activity of the unclarified sus—
pension in 0.35 M MgClz (Curve I. Figure 32-B). NaCl was
not as effective as MgClz. At 0.30 M NaCl. 55% of the phos-
phatase had been released from the particle but only 10%
more of the phOSphatase was solubilized even up to 0.8 M
NaCl. The ionic strength of either salt for 50% solubiliza-
tion of the phoSphatase was the same but solubilization above
that point did not coincide. PhoSphatase released seems to
be more dependent upon the divalent cation. The signifi-
cance of the increase in activity at high MgClZ concentra-
tions after the suSpension is clarified is not understood.
However it may be indicative of some control mechanism on

the enzyme by the particle.

Purification of the Starch Particle
3-P-Glycerate Phosphatase

In the isolation of the starch pellet by sucrose
density gradients the 3AP-glycerate phoSphatase on the
starch particles was enriched 46 fold. Further purifica-
tion of the starch particle 3aP-glycerate phoSphatase was
done using solubilized enzyme, because of the large amount
of information already gathered on the soluble 3-P-g1ycerate

phoSphatase from sugarcane. The starch particle enzyme was

172

Figure 32. Solubilization of the Particulate 3AP-Glycerate
PhoSphatase as a Function of Salt Concentration

To samples (0.10 ml) of the starch particles in 0.8 M
sucrose. 1 mM EDTA and 20 mM cacodylate buffer. pH 6.3. were
added 0.10 ml NaCl or Mg012 to give the final salt concentra-
tion. The samples were incubated for 30 minutes at 4°. Total
enzyme activity is shown for the suSpension (0——40) and for
the supernatant (A—A) after removing particles by centrifug-
ing at 10.000 x g for 20 minutes.

173

 

 

 

 

 

 

is
E IOOL'—.—.—'—-.—1—.—r-.—.—IIF-L
..x 1. Suspension/
x
0- 2 A K Supernatant,11
. Le". . . .1, .
3‘ O.l 0.2 0.3 0.4 0.5 0.6 0.8
[NaCl ]. M
x IOOng—O—OJ7‘CO:OTO-C .
Suspensioml

E ‘ "\ Supernatant. n

“c; 20— / 1
1 ““65 02 01.3 014 0%5 0.6
which L M

 

 

174
solubilized by sonification for 20 minutes at 4° in 0.8 M
sucrose, 20 mM cacodylate buffer, pH 6.3, and 1 mM ETTA.
After partial purification (Table XXII) of the particulate
(starch grain) phoSphatase the Specific activity was 10.75
(enriched 384 fold) compared to a Specific activity of 740
(enriched 2530 fold) for the most pure preparations of the
soluble phoSphatase from sugarcane leaves.

Fractionation by acetone precipitation was performed
in the same manner as that for the soluble sugarcane enzyme.
A volume of reagent grade acetone (-5°) equal to 40% of the
volume of the solubilized particulate 34P-glycerate phos-
phatase was added dropwise to the enzyme at 4°, the system
equilibrated for 20 minutes and then centrifuged for 10
minutes at 14,000 x g. The precipitate was discarded and
cold acetone, equal to 20% of the volume of the solubilized
enzyme, was added and the system equilibrated and centri-
fuged as before. The supernatant was discarded and the
precipitate resuSpended in 20 mM cacodylate buffer, pH 6.3,
and 1 mM EDTA. The recovery of the enzyme was 61% with
about a 7.5 fold enrichment.

The addition of 40 g of (NH4)2504 per 100 m1 of
acetone-fractionated enzyme precipitated about 25% of the
enzyme with no purification. Addition of more (NH#)280u
equal to 20 g per 100 m1 acetone-fractionated enzyme pre-
cipitated 34% of the enzyme with about 2-fold enrichment.

A summary of the purification of the particulate 3-P-glycer-
ate is presented in Table XXII. Further attempts to purify

1175

 

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176
the enzyme have not been made. Attempts to purify the par-
ticulate form of the enzyme as a particulate enzyme should
be considered as well as purification of the enzyme after
removal from the starch particle.

Further characterization of the starch-like particle
itself is a definite requirement. MicroscOpic examination
should be extended to electron microscopy, staining proce-
dures and chemical composition data. The distribution of
the starch particle must be examined eSpecially since it
was not found in all Spinach leaf preparations. At present
it is thought that the starch pellet could actually be
residues of the starch grains which are resistant to further
breakdown in the aged and stored Spinach leaves from which

the particles were isolated.

DISCUSS ION

3-P-Glycerate phosphatase from sugarcane leaves has
been purified 2530-fold with about 5% overall recovery. The
enzyme stoichiometrically cleaves 3-P-glycerate to produce
glycerate and inorganic phoSphate. The purification of the
enzyme was hampered initially by the phenol oxidases and
phenolic oxidation products as well as inefficient extrac-
tion procedures for tough sugarcane leaves. The use of
Polyclar AT, acid pH. EDTA and rapid acid fractionation were
the molt effective means of controlling the interference of
phenolic compounds. The acetone, (NH),)ZSOI+ and Sephadex
G-200 fractionation steps were optimized with regard to pH
and ionic strength so that most of the extracted protein
was removed along with probably all other phoSphatases,
since the relative Specificity did not change after the
0-200 gel filtration step (Table IV). The instability of
the enzyme alkaline pH and an isoelectric point of 6.8 pro-
hibited the use of anion excharge columns for purification.
Cationic exchange columns were most effective for further
purification. The P-cellulose column could be operated at
pH 4.0 with an additional 6-fold purification y_s 3-fold at
pH 4. 5. but recovery was very poor and afterwards the enzyme
was unstable even when the pH was adjusted to 5.9. The
multiple peaks of phoSphatase activity observed with DEAE-

177

178
cellulose or CM-Sephadex columns. It was felt that the
multiple peaking on DEAE-cellulose columns was caused by
cperating too close to the isoelectric point.

Stability
During purification the enZyme was stable if the pH

was kept below 7. Above pH 7 the enzymatic activity decayed
rapidly and irreversibly. Unlike P-glycolate phoSphatase,
BAP-glycerate phoSphatase did not require a tricarboxylic
acid for stability, and no tricarboxylic acids were found
bound to the enzyme as with P-glycolate phoSphatase. Pro-
longed dialysis against 1 mM EDTA or passage through.G-25
Sephadex did not inactivate the enzyme at any point in the
purification. It was concluded that no easily removable,
small molecular weight molecule is necessary for enzyme
stability. High ionic strength (NaCl or Na acetate) did

not effect the activity or stability, however for some
unknown.reason the enzyme was inactivated by (NHu)ZSOu pre-
cipitation after the 0-200 Sephadex filtration step. The
enzyme was reasonably stable for short times at room tem-
perature, both in the crude state and purified. The enzyme
was very stable at all states of purity at 4° and also
stable at -18° except in the (NH4)280u fraction. BAP-Glycer-
ate phOSphatase was rapidly inactivated at temperatures
above 40° with 50% loss of activity after 3 minutes at 50°
compared to only 10% loss of the tobacco leaf P-glycolate
phoSphatase activity at 75° (Randall and Tolbert, unpublished

results).

179
Eggymatic Properties

The sugarcane leaf BAP-glycerate phOSphatase is simi-
lar to other leaf phoSphatases in many of its biochemical
properties. Activity of the most pure enzyme was optimal
at pH 5.7 to 6.0 depending on the buffer systems, while
preparations of low purity had a pH optimum around pH 6.3.
This higher optimum with the crude extract may be more char-
acteristic of the in _v_i_y_o_ situation.

The apparent Km of 2.8 x 10"+ M for 3AP-glycerate
phoSphatase was similar to that reported for most other
leaf phoSphatases. There was no evidence for sigmoidal or
multiple enzyme kinetics. The enzyme was most active at
42° which is low compared to the optimal temperature of
P-glycolate phOSphatase at 70°. The effect of ionic
strength on the enzymatic reaction was negligible up to
about 0.5 M.

The Specificity of the enzyme was not absolute, but
it has been designated as 3eP-glycerate phoSphatase because
the rate of hydrolysis of BAP-glycerate was greater than for
all other substrates examined. BaGlycerol phoSphate, a
typical physiological substrate used for phoSphatases, was
hydrolyzed at 1/8 the rate of 3eP-glycerate. PEP was the
only physiological substrate which was hydrolyzed at a rate
greater than 501 the rate for 34P-glycerate. The use of
'p-nitrophenylphosphate (hydrolyzed at 66% of 3aP-glycerate
rate) as a substrate for phoSphatases is considered to be

of little value to understanding their characteristics.

180

3AP~Glyoerate is unlike the typical non-Specific acid phos-
phatase in.p1ants (99. 112) orԤ.,ggli (148), which hydro-
lyze most carbohydrate esters at about equal rates and at
lower rates than for the nonpphysiological substrate,
Ip-nitrophenylphoSphate. The plant acid phOSphatases dis-
cussed in the Literature Review usually had a preference for
hydrolyzing either 3'- or 5'-nucleotides. 3AP-Glycerate
phoSphatase however did not rapidly hydrolyze either 3'- or
5'-nucleotides nor did it show much preference for either.
The terminal pyrophoSphatase activity of the phoSphatase
was about 1/3 or less of that for 3éP-glycerate. ATP
hydrolysis at 37% of the rate BAP-glycerate was 4-fold
greater than the hydrolysis of pyrophoSphate. The enzyme
was inactive towards polyphoSphates and it did not have an
appreciable activity for converting NAIP(H) to NAD(H). The
diesterase activity, as measured by bisgp-nitrOphenylphos-
phate, was zero. however RNA or me were not tried as sub-
strates. The 3éP-g1ycerate phoSphatase was not able to
hydrolyze the CAP bond of three phoSphonic acid derivatives.

The Specificity of the enzyme showed no significant
change after the second acetone fractionation. The greater
than 3-fold increase in activity towards fructose-1,6-diphos-
phate after the first acetone fractionation could be
explained as a result of removing some inhibitor. However,
the enzyme lost Specificity. but not total activity during
aging, suggesting that some as yet unknown changewas occur-

ring in the enzyme. This phenomenon could be due to some

181
unfolding or proteolytic action on the enzyme thereby caus—
ing alterations in the properties of the substrate binding
site. Some constituent of the enzyme preparation might
also cause a conformational change in the enzyme which
would open or "loosen" the active site. Since the Specific-
ity at the various purification steps was not determined on
a preparation made in a very short period of time, it is
difficult to assess the changes in Specificity. Experiments
with mixed substrates and with various amounts of P-esters
indicated that the other phoSphophate esters, when present
with 34P-glycerate, were slightly inhibitory with regard
to total phoSphate released.

The experiments using mixed substrates gave no indi-
cation of mmre than one phoSphatase being present. The
isoelectric focusing of the 0-200 Sephadex fraction and the
most pure enzyme resulted in only one phoSphatase peak.

The Lineweaver-Burk plots indicated that only one enzyme
was present. These facts in addition, to the lack of

change in the relative Specificity during the latter stages
of purification. support the conclusion that one phoSphatase
was present.

It was not possible to demonstrate any cation require-
ment by the enzyme. Nine cation complexing reagents did not
significantly reduce the activity of 3AP-glycerate phoSpha-
tase. In fact, there was a Slight tendency for activity to
increase with these substances. The addition of various

cations did not stimulate or increase the phoSphatase activity.

182
Most divalent cations were inhibitory. eSpecially'an+, SnI+,
cu++. and 006+. Inhibition by Zn.'"+ has been used as a char-
acteristic feature of this particular phoSphatase, when com-
paring it to P-glycolate phoSphatase or alkaline phoSphatase
which are most active with Zn”. The salts of Pb” and Hg“
were typically inhibitory. The monovalent cations had no
effect and no anion effects were detected. With reapect to
divalent cations, BAP-glycerate phoSphatase is probably
more like the typical acid phoSphatases of‘g..ggl; and
yeast, which do not require cations for activity. It does
pose an interesting mechanistic question why two phoSphatases,
such as 3éP-glycerate phoSphatase and.P-glycolate phoSphatase
(requiring a divalent cation). each Specifically hydrolyzing
closely related substrates, can be so different.

The 34P-glycerate phoSphatase was not greatly
affected by alkylating agents. Only‘p-chloromercuribenzoate
at 10'3 M gave as much.as 24% inhibition. The phOSphatase
was typically inhibited by fluoride, L(+)tartrate and molyb-
date. The tartrate inhibition was competitive. L(-)tar-
trate and.meso-tartrate were not significantly inhibitory.

It appears that L(+)tartrate inhibits those phoSphatases
without cation requirements, perhaps suggesting some unique
nature of the active site. In contrast to P-glycolate phos-
phatase, cysteine and glutathione did not inhibit the 34P-
glycerate phosphatase. The tricarboxylic acids were also
slightly inhibitory.

Inhibitors of glycolate metabolism were ineffective

183
on 34P-glycerate phoSphatase activity. The use of a-hydroxy-
2-pyridinemethanesulfonate.$3;y£yg usually results in an
accumulation of glycolate by inhibiting glycolate oxidase.
This compound caused an accumulation of glycerate in photo-
synthesis experiments in sugarcane (74). However, our
attempts to find a glycerate oxidase in sugarcane or corn
leaves were negative.

A number of analogs of BAP-glycerate were investigated
in attempts to find an inhibitor of the enzyme which might
be usable for physiological studies, but none were found.

A number of metabolites of glycerate and BAP-glycerate in
addition to the products of the photosynthetic Cu-pathway
were not effectors of the phoSphatase. L—ASpartate at 10-3 M
provided a 10% stimulation of the phoSphatase activity.
PhoSphonic acid derivatives (Table IX) also produced a 10%
stimulation at saturating substrate concentration. The
reasons for the slight stimulation by L-aSpartate and the
phoSphonic acids are not known. One would reasonable expect
the phoSphonic acids to inhibit competitively. The stimula-
tion by L-aSpartate might be indicative of some means of

g 112 control since both 3-P-glycerate and L—aSpartate are
major, initial products of Gag-fixation in Cg—plants.

The kinetics of inhibition by glycidoléP were typical
of competitive inhibition. but no phoSphate was released
from glycidoléP. The enzyme was irreversibly inhibited by
glycidolAP. .Alkylation of the active site by the glycidol
or epoxide moiety must prevent the enzyme from completing

184
the hydrolysis, thus, this compound appears to bind at or
near the active site. The inhibition and alkylation may
indicate a similarity of enzymatic mechanism for the phos-
phatase and for triose isomerase and enolase reactions

which are also inhibited irreversibly by glycidoleP (135).

wsical Properties

3AP-Glycerate phoSphatase is quite large. It was
slightly retarded on.G-200 Sephadex with a 1.4 to 1 ratio
of the elution volume to void volume. from which a molecular
weight is estimated to be in the ISO-200.000 range. The
sucrose density gradients with low ionic strength produced
evidence of a range of active forms or aggregating forms.
The system.was probably in a rapidly associating-dissociat-
ing state, and the most prevalent form.was around 13 S or
around 300,000 deltons. Increasing the ionic strength to
0.25 M with KCl resulted in only one form of the enzyme
with a 8.0 S value or around the 160.000 molecular weight
range. This latter value is in agreement with the estimate
from the 6.200 Sephadex column. Whether or not the multiple
aggregates or the associating-dissociating phenomenon is
significant is difficult to predict at this time. The
phenomenon could be due to low protein concentrations or
some interferring protein that has not yet been removed.
The tendency to aggregate may conceivably have physiological
significance. lg;ygyg_aggregation could control the rate

of hydrolysis of the substrate, although the enzyme in the

185
various forms was active. Some undetected factor may also
be necessary for such a control mechanism. The diurnal
variations in the enzyme (discussed below) could possibly
be a manifestation of this aggregation phenomenon. one form
predominating during the more active period for the enzyme.
The effects of various metabolites on the aggregated forms
has not been examined.

The SUB-polyacrylamide electrophoresis experiment
produced two polypeptide bands from the most pure enzyme
preparation. The major band had an approximate molecular
weight of 51,000 and the minor band was about 62,000. The
molar ratio of the two bands was 4.3 to 1. If both of these
were subunits, it is difficult to imagine a combination of
these two units that would give a molecular weight in the
140-200.000 range. One polypeptide could be a contaminant.
If this were the case, one would have a tendency to pick
the 51,000 unit chain as the phOSphatase polypeptide. This
would suggest an enzyme of 3 or u polypeptide chains. If
the smaller peptide chain was the result of cleavage of a
10-11.000 molecular weight peptide from 62,000 pieces. one
wonders at the incompleteness of the process. Non-identical
'subunits in a ratio of h to 1 or 3 to i is not impossible,
but an odd number of subunits (h to 1) would be very unusual.
The fact that the enzyme will not migrate on regular poly-
acrylamide gel electrophoresis (5% gels) can be considered
to support the idea that the enzyme is quite large (aggre-
gated) at low ionic strength. In the three systems tried

186
there were no detectable protein bands on the gels. even
though standards were easily detected. The lack of detect-
able protein on the gels indicates that the BAP-glycerate
phoSphatase preparation was probably quite pure but not
necessarily homogeneous. This suggests that two small
polypeptide bands in the SDS-polyacrylamide were from one
enzyme or the contaminating protein was bound closely to
the phosphatase.

Eggsiological Considerations

The results of the survey for the phoSphatases showed
that the BAP-glycerate phosphatase was most active in Cu-
plants but was also found in significant amounts in 03-plants.
The rate of BAP-glycerate hydrolysis in C3-p1ants was 0.7 to
9 umoles per mg chlorophyll per minute as opposed to 002
fixation rates of 1 to h umoles 002 per mg chlorophyll per
minute (16). Thus there is enough BAP-glycerate phosphatase
even in C3-plants to hydrolyze most of 3-P-glycerate formed.
In the 04-p1ants the enzyme is consistently 2- to 3-fold
more active than the 03-plants and still as great as, or
greater than the rate of photosynthesis. These facts support
the idea that the BAP-glycerate phosphatase is closely
involved in the photosynthate metabolism in 04-plants.

The enzyme seems to be localized in the leaf tissue
and is 20-fold more active there than in the stem and root.
The very low levels of BAP-glycerate hydrolysis by extracts
of stem.and roots can easily be attributed to acid phospha-
tases of these tissues. Limiting the enzyme to leaf tissue

187
does not necessarily link the enzyme to photosynthesis.
Therefore the specific location in the leaf may help deline-
ate its function. As described in the Literature Review,
sugarcane has two types of chloroplast containing cells,
the mesophyll cells and parenchyma or bundle sheath cells.
Appendix A presents the results of experiments that con-
cluded that the BAP-glycerate phosphatase is mainly in the
mesophyll cells as compared to the bundle sheath location
for the P-glycolate phosphatase. The mesophyll cells con-
tain the chloroplasts with enzymes unique to the Cu-pathway
of 002 fixation (Table 1). Different lines of evidence
were presented to indicate that the phosphatase was not in
any particulate fraction from sugarcane leaves. Chloroplast
isolation procedures specifically designed for sugarcane
leaves were unsuccessful in localizing the 3-P-glycerate
phosphatase with the.mesophyll chloroplasts. Other differ-
ential centrifugation procedures in buffered, isotonic media
failed to locate the enzyme with peroxisomes or any other
subcellular body. These results contrast with those of
Matile gtflgl. (149) who have presented evidence that there
is considerable acid phosphatase in spheroscmes or dictyo-
somes of higher plants.

Non-aqueous density fractionation procedures located
the BAP-glycerate phosphatase in the non-chloroplastic
fractions of the leaf, in complete agreement with the acid
phosphatase distribution in sugarcane leaf tissue report by
Slack 3.? 21.. (#1). This does not necessarily mean that the

188
enzyme is not involved in the pathway of carbon metabolism
during photosynthesis. There is increasing evidence (5#,
57, 150) that the flow of carbon between the chloroplasts
of the mesophyll cells and chloroplasts of bundle sheath
cells is very rapid and obligatory to the total photosyn-
thetic process. PEP carboxylase is believed to be located
in the peripheral reticulum of the mesophyll chloroplasts
for easy access to the 002 and to facilitate the transport
of oxaloacetate or malate to the bundle sheath chloroplasts.
Triose isomerase and glyceraldehyde-P-dehydrogenase are in
both types of chloroplasts. BAP-Glycerate, the product of
the "transcarboxylation reaction," is distributed about
evenly between the two chloroplast types. These facts all
lend support to rapid movement of carbon compounds between
the cells. A phosphatase could conceivably regulate the
direction of carbon flow or create a concentration gradient
of a particular compound in a metabolic sequence.

Further evidence for the involvement of the BAP-glyc-
erate phosphatase in photosynthesis or in related metabolism
in eggplants is supported by its formation during the bio-
genesis of the chloroplasts. The enzyme activity increased
10-fold on a fresh weight basis and h—fold on a protein
basis after etiolated tissue was illuminated. Slack'gg‘g}.
(151) reported very little change in acid phosphatase levels
in etiolated corn and sorghum.leaf after exposure to light
but the enzymes involved in photosynthesis increased h- to
12-fold. The increase in BAP-glycerate phosphatase activity

189
during greening of etiolated tissue is the same phenomenon
as increases in P-glycolate phosphatase activity (76),
which is found on or in the chloroplasts. The evidence
supports a function for BAP-glycerate phoSphatase in the
photosynthesis of Cu-plants.

§peculations on the Function(s)

The exact function of BAP-glycerate in leaves is not
known. Since this phosphate ester is a primary product of
photosynthetic C02 fixation and a key intermediate in
glucose synthesis and photosynthate metabolism, numerous
regulatory functions for the phosphatase can be invisioned.

Regulation of the photosynthetic carbon pathways:
Certainly any hydrolysis of 3-P-glycerate as it is formed
during 002 fixation will curtail the rate of photosynthesis
by slowing the regeneration of RuDP. BAP-Glycerate also
could exert product inhibition on the RuDP carboxylase;
thus, hydrolysis of BAP-glycerate in this case could epped-
up photosynthesis. Whether the phosphatase functions for
such regulation in 1.312 he not been tested. It is known
that during short-term luCoz photosynthesis by many plants,
considerable carboxyl-labeled glycerate is formed.

Serine formation: P-Glycerate and glycerate are
metabolic precursors for serine formation in plants. Aside
from synthesis in the glycolate pathway, evidence supports
the predominance of the nonpphosphorylated pathway of serine
synthesis in green leaves. Serine, besides being an essen.
tial amino acid, can be a precursor of glycine and C1 syn-

190
thesis. A major flow of carbon to the C1 pools must occur
during plant growth.

Regulation of starch synthesis: 3-P-Glycerate is an
allosteric activator of Arr-glucose pyrophosphorylase (152)
which forms the substrate the starch synthetase. Regulation
of the pool size of BAP-glycerate should have a profound
influence on the rate of starch formation.

Carbon transport: BAP-Glycerate phosphatase may
function as part of a transport mechanism for photosynthate
between the two different cell types in leaves of Cu-plants
such as sugarcane. Several additional factors must be
considered for such a function.

(1) BAP-Glycerate phosphatase is generally more

active in Cu-plants than in C3-plants.

(2) BAP-Glycerate phosphatase and glycerate kinase
in Cu-plants are primarily located in the meso-
phyll cells, along with the enzymes for the Cu-
pathway of C02 assimilations (Table 1).

(3) According to Andrews (5“) the "transcarboxylation"
reaction and BAP-glycerate should be formed in
the bundle sheath chloroplasts where there is
inadequate reducing power.

(#).At least half of the BAP-glycerate produced by
"transcarboxylation" in Cupplants is found in
the mesophyll cells along with about half of the
NAIF -glyceraldehyde-P -dehydrogenase and triose

isomerase (#1).

191

The BAP-glycerate could be moving to the mesophyll
cell and/or chlorOplast for further reductions or metabolism
to regenerate the primary C02 acceptor (PEP) for the Cu-
pathway. With.regard to this, I would like to postulate
that BAP-glycerate phosphatase may be a part of a permease
or transport system to move carbohydrate from the bundle
sheath cells into the mesophyll cells or mesophyll chloro-
plasts. This scheme is represented in Figure 33 in combin-
ation.with.part of the data from Hatch and Slack (16) con-
cerning the two photosynthetic pathways in different types
of cells. BAP-Glycerate phosphatase would be part of a
facilitative diffusion process by hydrolyzing BAP-glycerate
thereby creating a natural diffusion gradient for carbon
flow towards the mesophyll cells and chloroplasts. The
concentration of BAP-glycerate, an inhibitor of RuDP car-
boxylate, would be kept low in the bundle sheath preventing
inhibition of the carboxylase, and the carbon would be
moved to the location of adequate reducing power in the meso-
phyll cells.

If the mesophyll chloroplasts are more permeable to
glycerate than 3éP-glycerate, the phosphatases would be
facilitating transport into the chloroplast as well as the
cell. Glycerate in the mesophyll chloroplast would be
phosphorylated by the glycerate kinase located in the
chloroplast and then reduced and isomerized to DEAF. The
IEMP would then be available for conversion to the other
intermediates of the Cuppathway. Hatch and Black (16) have

192

Figure 33. Proposed Intercellular Hove-ent of New Fixed Carbon
Between Bundle Sheath and Heeophyll Cel

 

 

 

 

 

 

 

 

 

HESOPHYLL CELLS BUNDLE BREATH CEIlS
C02 II
II
0AA = Malate iii ; Malate NADP*< \
H
II
II
AMP ATP n NADPH
PEP (—>—<—— Pyruvate ‘4 yr Pyruvate
H
’\ PP P
i l H
\ H [002]
ll RuDP
II
\ ATP P1 :1 I‘ ., ADP
l
3-P-Glycerate {-4- Glycerate 3-P-Glycerate \— ATP
II
AT? H 3-P-Glycerate
H
AIP ll ATP
H
II ADP
1.3-d1-P-Glycerate H
H
NAIPH ll 1 ,3-d i-P -Glycerate ‘
ll < ‘ j
w ~ C C
H \L > j
H
3‘Pfilyceraldeh 4* \ [J
yde (—-> DHAP .r I DHAP 3-P-Glyceraldehyde
H V
H C6-P CS-P
ll

:: A3 \> .... .7... /’

I I . Sucrose Starch

193

suggested on the basis of other data that DHAP could be a
major transport form of carbon from the mesophyll cells to
the bundle sheath cells.

Substitute forgglycolate pathggy ingperoxisomes:
DeDuve (153) has emphasized that peroxisomes occur in tissue
capable of gluconeogenesis, though how the particle functions
in this manner is not known. BAP-Glycerate phosphatase in
the Cueplants could be a substitute for a gluconeogenic
function of the glycolate pathway or plant peroxisomes.
Glycerate is the major product of the glycolate pathway in
leaf peroxisomes and the glycerate is subsequently converted
to sucrose. Through the action of the BAP-glycerate phos-
phatase, photosynthate can be converted directly to glycer-
ate. In effect the phosphatase has bypassed the glycolate
pathway and the loss of 251 of the carbon as 002 via peroxi-
somal.respiration and the hydrolysis of an extra phosphate
ester, since two P-glycclate molecules must be hydrolyzed to
make one glycerate. If glycolate metabolism or peroxisomal
metabolism.is essential for photosynthesis and polysaccharide
formation in C3-plants, then BAP-glycerate phosphatase could
be serving this same essential function in a more efficient
manner for Cir-plants. The reciprical relationship between
34P-glycerate and P-glycclate phosphatases in the soybean
varieties also would support BAP-glycerate phosphatase as a
substitute for the glycolate pathway as a glycerate forming
system. The total possible glycerate formation by both

systems is about the same among the 15 soybean varieties.

19#
Diurnal Variations in Activity

The diurnal variations of the phosphatase activity
may also be reconciled with an enzyme involved in photosyn-
thesis. Phosphatase activity increased 50% in the latter
part of the daylight when the photosynthetic activity has
decreased. Even at the periods of lowest BAP-glycerate
activity, this activity was as high as the PEP-carboxylase
activity reported by Ratch.and Slack (16). The 50% increase
in the phoSphatase activity late in the afternoon could be
indicative of its role in regulating the metabolic fate of
photosynthetic carbon pools.

There are several possible implications of the diurnal
variation in the 3-P-glycerate phosphatase activity. During
the waning hours of daylight the plant could be recalling
carbon from storage (i.e. assimilatory starch) for further
metabolism.for maintenance, growth and additional tranSport.
The assimilatory starch is mainly located in the bundle
sheath chloroplasts and when the carbon from this starch is
reassimilated into the metabolism of Cu-plants, the soluble
phosphatase could be instrumental in creating a gradient for
BAP-glycerate flow from the bundle sheath cells to the meso-
phyll cells. This flow of BAP-glycerate out of the bundle
sheath chloroplasts would also reduce the concentration of
BAP-glycerate and in turn starch formation should be our-
tained. The mesophyll cells must also have a large portion
of the enzymes for further metabolism of the carbon from

starch, to such metabolites as pyruvate or acetyl 00A for

195
the Krebs cycle and lipid synthesis.

Glycerate is also the major product of the glycolate
pathway and assuming that there is significant metabolism
through this pathway in Cupplants, the condition favoring
glycolate biosynthesis decrease in the later part of the
day. Thus it is reasonable that the need for glycerate
formation could be covered by increased hydrolysis of 34P-
glycerate. Since the glycerate from the glycolate pathway
is probably metabolized to sucrose (73), the increased
phosphatase action late in the day would also permit sucrose
formation and continue the drain of carbon out of the photo-
synthetic fixation cycles.

Photosygthetic Efficiency

Photosynthesis is obviously a major determinant of
crop yield. Crop species with the highest rates of photo-
synthesis (sugarcane, sorghum) are also the highest yield—
ing species. Photorespiration or peroxisomal respiration
(82), which does not appear to be coupled to any energy
conserving mechanism, reduces the net photosynthetic effi-
ciency of a plant. Consequently the efforts of investiga-
tors attempting to use C02 fixation rates as an index of
yield potential are often inaccurate. P-Glycolate phospha-
tase provides the substrate for photorespiraticn and there-
fore has the potential of being the limiting enzyme.
Glycolate oxidase, as shown by Curtis _e_t 2;. (1&7), is
probably not a limiting enzyme in photorespiraticn.

196

The results of our survey for the two phoSphatases in
various plants appeared sufficient to support conclusions
that these two enzymes could be indicative of photosynthetic
efficiency. 34P-Glycerate phosphatase appears to be more
important to the metabolism of the photosynthate of Ch‘
plants than 03-plants. It is reasonable to believe that
the two phosphatases are reciprocally related in function
in the two types of plants. The BAP-glycerate phOSphatase
could be considered a marker for the more efficient photo-
synthesizing plants or plants having lower levels of C02-
photorespiration. These conclusions coupled with the evi-
dence that BAP-glycerate phosphatase action may substitute
for the glycolate pathway were the basis for experiments
with the soybean varieties. Soybeans have high levels of
activity for both phosphatases.

The soybean plant is a typical C3-plant with photo-
respiraticn which supposedly makes it less efficient. The
hypothesis was that BAP-glycerate phosphatase would be a
marker for more efficient soybean plants. The relative
activities of the phosphatases might provide a biochemically
based index for yield potential of a plant in early stages
of varietal development by the plant breeder.

The results of investigations on the 15 soybean
varieties were the opposite to those predicted. With
increasing photosynthetic rates the level of P-glycolate
phosphatase activity increased and 3AP-glycerate activity

decreased. Hopefully, this correlation may yet provide a

197
biochemical index for photosynthetic potential. Efforts to
substantiate the correlation are hampered by lack of fully
established functions for both glycolate metabolism and the
34P-glycerate phosphatase.

The results of this soybean varietal experiment are
inconclusive by itself but it does provide a basis for fur-
ther experiments such as similar assays for the peroxisomal
enzymes. The relative levels of the phosphatases may pro-
vide a rapid enzymatic assay for determining relative
photosynthetic or growth efficiency. Dr. W: L. Ogren at the
University of Illinois is pursuing this idea, and from other
physiological variations he thinks there is a positive cor-
relation between growth and amount of peroxisomal activity
(personal communication). The function of P-glycolate
hydrolysis (glycolate pathway) versus 3eP-glycerate hydrol-
ysis and the differences in subsequent metabolism are confus—
ing. But if the differences in metabolism that result can
be linked to grain output or composition, an additional
method can be added to the means of evaluating new varieties.
There is precedent for linking enzyme levels with grain out-
put. Bageman 33 §_]_.. (154) have found that the level of
nitrate reductase in the wheat leaves is positively corre-
lated with protein levels in the grain of the wheat.

Discussion of the Particulate géP-Glycerate Phosphatase
Investigation of the particulate or starch grain 34P-
glycerate phosphatase is being continued. The biochemical

198
properties determined to date are essentially identical to
those of the soluble sugarcane BAP-glycerate phosphatase.
The known.properties of the particle itself are limited.
The particle is probably polysaccharide in nature in that
its density is greater than 2.3 M sucrose and because 8-
amylase released reducing sugars concomitant with the
release of the enzyme. The binding of the enzyme is likely
to be ionic, since the high ionic strength solubilized it.
But the high ionic strength also could possibly be instru-
mental in.p1asmalyzing some closed membrane system. Either
possibility could be reconcilable with the fact that the
enzyme can also be solubilized by extended sonification.

The question still to be answered is whether or not
the particulate form of the enzyme is of physiological origin
or an artifact of isolation such as non-specific binding.

If the particulate form of the enzyme is of _i_n 133.9. origin.
its possible function(s) is highly speculative.

A possible function of the particulate 3eP-glycerate
phosphatase is an involvement with the regulation of starch
synthesis or degradation. Such a function is based upon
reports that the control of the biosynthesis of assimilatory
starch in spinach leaves is mediated by BéP-glycerate (152).
Arr-Glucose pyrophosphorylase, the enzyme which makes the
substrate for the starch synthetase. is an allosteric enzyme
activated 50-fold by 2 x 10-5 M BAP-glycerate (152). The
BAP-glycerate concentration thus becomes the fine control

on starch synthesis. If the concentration drops due to

199
curtailment of photosynthesis which provides the 3-P-g1ycer-
ate and ATP. starch synthesis stops and will not begin until
the pools of the intermediates of the carbon reduction cycle
are full. Hydrolysis of 34P-glycerate by the phosphatase
would lower the concentration of BAP-glycerate, inactivate
the synthetase and provide P1 for a phosphorylase degradation
of starch. Inorganic phosphate also inhibits the ALP-glucose
pyrophosphorylase (152). The effect of BAP-glycerate on
starch.phosphorylase is not known. Whether or not the par-
ticulate BAP-glycerate phosphatase does function in this
manner to control starch formation and degradation is
admittedly Speculative at this time. For plants there is
lack of knowledge with regard to regulatory mechanisms and
intermediates such as cyclic AMP. Key phosphate esters such
as BAP-glycerate and specific phosphatase and kinase sys-
tems could possibly be a manifestation of the plant's regu-
latory requirements.

Best success in isolating the starch-like particles
with phosphatase activity was obtained with stored spinach
leaves in which starch.rich.particles should have been
depleted. Normally destarching of leaves is accomplished by
3 days of darkness. Little is known about any residual
particle with primer starch and the necessary enzymes for
starch synthesis. As isolated from old stored leaves the
particle carrying the phosphatase could be the primer or
limit dextrin of the starch grain or the membrane surround—
ing the starch grain or both. Regulation of the phosphatase

200
may involve the physical properties of particle, and perhaps
the enzyme is inactive‘in vivo in either the bound or

soluble form.

SUMMARY

3-PaGlycerate phosphatase from sugarcane leaves was
isolated and purified 2530-fold. The purity of the enzyme
is uncertain, but no protein bands could be detected using
three different sets of electrophoresis conditions. The
enzyme stoichiometrically hydrolyzes D-3eP-glycerate to
Ibglycerate and inorganic phOSphate. The enzyme was not
absolutely specific, but it was at least 2—fold more active
with.34P-glycerate than most other substrates. PEP was
hydrolyzed at 0.66 of the rate of BAP-glycerate. Optimal
enzymatic activity was between pH 5.7 and 6.0. The enzyme
required no divalent cations or other detectable cofactors
nor was it inhibited by EDTA. The apparent Michaelis COD!
stant for 3éP-glycerate was 0.28 mM. The purified phospha-
tase had an isoelectric point at about pH 6.8 and did not
electrophorese in 3 different buffering systems and pH'S.
The enzyme was stable at -18° or 4° indefinitely, and at
room temperature for brief periods. Over 50% of the enzyme
was inactivated by incubation at 50° for 3 minutes.

The 3eP-glycerate phosphatase was inhibited by
typical phosphatase inhibitors, L(+)tartrate, molybdate
and fluoride.. GlycidoleP inhibited the enzyme irreversibly
without concomitant release of inorganic phosphate. Three
phosphonic acid derivatives, phosphonoacetate, 2-amino

201

202
phosphonoacetate and 2-amino-3-phosphonopropionate were not
hydrolyzed by the enzyme but stimulated (10$) the enzymatic
activity with substrate at saturating concentrations.
LnAspartate stimulated the phosphatase to a similar degree
as the phosphonic acids.

The enzyme showed an.aggregation phenomenon on
sucrose density gradients of low ionic strength. On high
ionic strength sucrose gradients, the enzyme centrifuged in
one form at approximately 8.0 S, with an estimated molecular
weight in.the 160.000 unit range. SDBAPolyacrylamide elec-
trophoresis yielded a major band at 52.000 molecular weight
units and a minor band at 62,000. The molar ratio of the
two bands was 3.6 to 1.

The enzyme is located in the leaf tissue of sugarcane.
[Afferent lines of evidence localized the sugarcane 3-P-
glycerate phosphatase in the cytoplasm of the mesophyll cells
and there was no evidence of the sugarcane enzyme being par-
ticulate. The enZyme activity increased at least a fold on
a protein basis and 10 fold on.a chlorophyll basis during
the greening of etiolated tissue. On sunny days the enzyme
activity displayed a diurnal variation with at least a 50%
increase in activity during the late daylight hours and early
darkness.

The BAP-glycerate phosphatase was generally more
active in plants with the Cu-pathway of C02 assimilation but
the activity in C3-plants was enough to account for any
glycerate formed. P-Glycolate phosphatase was generally

203
most active in plants with the C3-pathway of 002 assimilation
but significant and sufficient activity was found in Cu-plants
to account for any glycolate formed. The levels of the
activities of P-glycolate phosphatase and 3éP-glycerate phos-
phatase were correlated positively and negatively respectively
to the rates of 002 fixation in 15 varieties of soybeans
(Glzgine'geg. L. Merrill). These phosphatases may be used
as indices of photosynthetic potential.

A particulate or starch grain 3-P-glycerate phospha-
tase from.spinach leaves was discovered and partially char-
acterized. The enzyme was pelleted through the 2.3 M or
2.5 M sucrose layer of discontinuous sucrose gradients used
for preparation of peroxisomes. Because of its density, a
positive reaction of the material to KI-Ig reagents and the
release of the phosphatase and reducing sugar by B-amylase,
the pellet and particles to which the enzyme was bound were
tentatively termed the starch pellet and starch particles.

The biochemical characteristics of the particulate
34P-glycerate phosphatase were similar to the soluble
enzyme from sugarcane leaves. Optimal activity was around
pH 5.8 and it required no divalent cations or other cofac-
tors. The enzyme exhibited normal hyperbolic kinetic plots
with an apparent Michaelis constant of 9 x 10"“ M for 3-P-
glycerate. The phosphatase was not absolutely specific but
hydrolyzed BAP-glycerate about 1.5 times faster than the
other physiological substrates. The particulate phosphatase

was enriched #6-fold over the crude spinach extracts.

20h
Solubilization of the starch particle 3eP-glycerate
phosphatase was possible by extended sonification treatment,
0.35 M MgClz or incubation with B-amylase. Passage through
a French pressure cell. homogenization in a.Potter—Elvjehm.
repeated freezing and thawing, changing the pH and extended
hydrolysis were ineffective inreleasing the enzyme from the
particles. The solubilized BAP-glycerate phosphatase from
the starch particles was partially purified by acetone and
(NH4)2304 fractionation. The partially purified enzyme was
enriched 38h fold with a Specific activity of 10.8 and a 3h%

recovery.

10.

11.
12.

13.

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APPENDICES

APPENDIX A

ENZYMES OF THE GLYCOIATE PATHWAY IN PLANTS WITHOUT C02-
P HOTORESP IRAT ION

D. W. Rehfeld, D. D. Randall, and N. E. Tolbert

Canadian Journal of Botany, Vol. 48, p. 1219.

215

APPENDIX B

 

 

 

 

 

 

 

 

 

 

Plant Variety
Cotton Gossypium hirsatum L. ---
Tobacco Nicotiana tabacum L. Maryland Mammoth
Spinach Spinacia L. Longstanding

Bloomsdale

Sunflower Helianthus L. Mammoth Russian
Wheat Triticum vulgare L. Thatcher
Alfalfa Medicago sativa L. ---
Tomato Hoppersicon Mill. Big Boy
Bean Phaseolus vulgaris L. Sonalac

--- Atriplex Eatula m ---
Sudan grass --- Piper
Sudum ..-- --..
Sorghum Sorghugirgalepense (L.) ---
Pigweed Amaranthus hybridus (L.) ---
Sugarcane Saccharum CL 41-223
Corn _Z_eg 9212 L. Michigan 500

--- Atriplex £3332 ......
Crabgrass Digitana sanquinalus -..-
Burmuda Cmodogrgactzlon (L.) ---
Bluegrass 1192 cogpressa L. ---
Bent Grass Aggostis tenuis ---

216

Plant

 

Merion bluegrass
White oak

Red maple
Mountain ash
Cottonwood
American elm

Red spruce

217

Poa pratenis
Querus alba L.

522; rubrum L.

Sorbus americana marsh
Pcpulus‘ggl toides marsh
211mg americana L.

Picea rubens saxy

Variety