METABOLISM OF GLYCGLZC ACiD

floats for 1410 Dear» a! Ph; D.
MICHIGAN STATE UNIVERSITY

Myron Kuczmk
1961

 

 

0469

This is to certify that the

thesis entitled

METABOLISM OF GLYCOLIC ACID

presented by

Myron Kuczmak

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Biochemistry

firm

Major professor
N. E. Tolbert
Date August 5: 1961

LIBRARY

Michigan State
University

 

 

METABOLISM OF GLYCOLIC ACID

By

Myron Kuczmak

A THESIS

Submitted to the School for Advanced Graduate Studies
of Michigan State University in partial
fulfillment of the requirements for
the degree of

DOCTOR OF PHILOSOPHY
Department of Biochemistry

1961

ACKNOWLEDGEMENT

The author wishes to express his sincere
appreciation to Dr. N. E. Talbert, under whose
direction this investigation was accomplished,
for his inspiration, guidance, patience, and
understanding during the course of investigation

and preparation of this thesis.

ii

METABOLISM OF GLYCOLIC ACID

By

Myron Kuczmak

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies
of Michigan State University in partial
fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1961

Myron'Kuczmak

ABSTRACT

The purpose of this investigation was to describe the
conditions for activation of glycolic acid oxidase, both.;g
‘ziggg and‘in‘zizg and to study the inhibitory effect of
glycolate and glyoxylate on citric acid cycle oxidation.

Glycolic acid oxidase, an enzyme, catalyzing the
oxidation of glycolic acid to glyoxylic acid and H202 and
also glyoxylic acid to oxalic acid is a flavoprotein with a
prosthetic group of FMN. FAD cannot substitute for FMN when
the enzyme is partially purified. Utilization of FAD by
crude saps for enzyme activity was due to enzymatic con-
version of FAD to FMN.

Glycolic acid oxidase is very active in extracts from
leaves of green plants. However, extracts of leaves from
etiolated wheat seedlings and sheaths of both green and
etiolated plants contain much less activity. Extracts of
etiolated leaves contain apoenzyme which.eould be activated
by 10‘4 M FMN. The amount of apoenzyme was considerably
less than the amount of enzyme in green tissue.

The enzyme is predominantly a soluble cytoplasmic
enzyme, although some enzymatic activity is associated with
chloroplast or mitochondria. During the oxidation of glycolic
acid by mitochondria there was a little esterification of

crthophosphate.

iv

Exposure of etiolated plants to the light causes the
formation of both active enzyme and of more apoenzyme.
Etiolated plants contain proportionally more apoenzyme to
holoenzyme than plants exposed to light. The ratio of apo-
enzyme to holoenzyme in etiolated wheat seedlings is 9 to 1
and in green ones about 5 to 1. The amount of the apoenzyme
plus holoenzyme from etiolated plants is much less than the
amounts in green plants. Thus the increase in this enzyme
is associated with greening of the plant.

Green plants contain twice as much FMN as etiolated
plants, but this physiological level of FMN was about 10'8 M,
which is below the 10‘4 M concentration of FMN necessary to
activate the enzyme ig‘yiggg.

Glycolic acid oxidase can be activated ig,zit§g at
2°C by the incubation of plant cell-free homogenates with
its substrates, glycolate, lactate or aL-hydroxybutyrate.
This method of enzyme activation cannot be duplicated on a
dialyzed ammonium sulfate precipitate of the enzyme. The
nature of the igmlgtgg activation is not known.

The enzyme also can be activated iglzizg in the dark
by feeding the etiolated plants with glycolate, lactate,
az-hydroxybutyrate or glyoxylate.

Both glycolate and glyoxylate are the effective
inhibitors of the oxidation of citric acid cycle acids by

isolated plant mitochondria. Because glycolic acid is

vi

rapidly oxidized by pea mitochondria by associated glycolic
acid oxidase, the inhibitory effect of glycolate was attri-
buted to the glycxylate formed. Glyoxylate was active as an
inhibitor of the oxidation by the mitochondria of each acid
from the citric acid cycle. The 2/0 ratio of residual
activity in the presence of the inhibitor was not reduced in
the inhibited system. Glyoxylate also severely inhibited the
action of isolated malic and lactic dehydrogenase.

The inhibitory effect of glyoxylate can be reversed
by addition of excess of DPN. This was demonstrated with
both malic or lactic dehydrogenase and with mitochondrial
oxidation.

The inhibition by glyoxylate of citric acid cycle
oxidation is probably due to non-enzymatic formation of a
complex with reduced DPNH. This complex was detected on

paper chromatograms by its fluorescence and Rf values.

TABLE OF CONTENTS

INTRODUCTION . .

LITERATURE REVIEW

Glycolic Acid in Microorganisms

Glycolic Acid in Animal Tissues . . . . . . . . .

Glycolic Acid in Plants . . . . . . . . . . . . .

GlyC011c ACid OXidaae o e e e e e e e e e e o e 0

Role of Glycolic and Glyoxylic Acid in Plant
Respiration . . . . . . . . . . .

O O O O O O 0

MATERIALS AND METHODS

O
O
O
O
O
O
O
0

Plant Material 0 e e e e e e e 0
Preparation of Tissue and Enzyme
Assays and Analyses . . . . . . .
Source of Chemicals .

O O O O
O O O 0
O O O O
o O O O
o 00 0

RESULTS AND DISCUSSION .

A. GLYCOLIC ACID OXIDASE IN PLANTS . . . . . . .
Organ and Cellular Distribution of
Glycolic Acid Oxidase . . . . .
Activation of the Enzyme by Added Excess
Of FM 0 O O 0 O O O O O O O O O O 0
Effect of Light plus FMN on Enzyme
ACLiVity e e e e e e e e e e e e e e e e
Flavin Content of Wheat Leaves . . . . . .
Relative Contents of Apoenzyme in Green
and EtiOlated Tissues e e e e e e e e e 0
Activation of the Enzyme from Ammonium
Sulfate Precipitate . . . . . . . . . .
Activation of the Enzyme in vitro by
Incubation with the Substrate . . . . . .
Substrate Activation of the Enzyme i;
V170 e e e e e e e e e e e e e e e e e e
B.

RESPIRATION OF PLANT MITOCHONDRIA

General Experimental Condition Affecting
Respiration . .

C O O O O O O O C O O O O

Page

d

-0\i.~u U

18
18
18
20
22
24

24

viii

Page

Oxidation of Glycolate and Glyoxylate
byMitOChondrla............. 7‘
Inhibition of Oxidation by GlyColate
or Glyoxylate . . . . . . . . . . . . . . 72
Consideration of ”Oxalomalate" as
Inhibitor.........~....... 81
Oxaloacetic Acid and oL-Ketoglutaric
AC1d OXIdation e e e e e e e e e 85
Stimulation of Succinate Oxidation by
Glyoxylate and Inhibition by Thic-
81y0013teeeeeeeeeeeee O
Stimulation of Glyoxylate and Succinate
OfldatlonbyFMNcocc........ 90
Interreactions Between Succinate
Glyoxylate and Glutamate . . . . . . . . 93
Effect of Glyoxylate and Cysteine on
Mitochondrial Respiration . . . . . . 93
Effect of Tyrosinase and Ascorbic Acid
Oxidase................. 95
In vivo Inhibition of Respiration by

. 87

“Glyoxylate.......... ace. 98
Nature of the Glyoxylate Inhibition . . . . 98

Reversal of the Glyoxylate Inhibition
byDPNOOOOOOOOOOOOOOOO.102
SUMMARY 115
117

BIBLIOGRAPHY O O O O O O O O O O O O O 0 O O O O O O O

I.

II.

III.

IV.

V.

VI.

VII.

VIII.

IX.

X.

XI.

XII.

XIII.

XIV.

XV.

TABLES

Activity of glycolic acid oxidase from
Plant tissues 0 e e e e e e e e e e e e e e

Glycolic acid oxidase from shoots and roots
of green pea seedlings . . . . . . . . . .

Glycolic acid oxidase in leaves and sheaths
of Thatcher wheat . . . . . . . . . . . . .

Location of glycolic acid oxidase in
fractions from Swiss chard . . . . . . . .

Location of glycolic acid oxidase in
fractions from etiolated Thatcher wheat . .

FMN activation of glycolic acid oxidase
from green and etiolated plants . . . . . .

Oxidation of glycolate and glyoxylate by
etiolated barley sap . . . . . . . . . .g.

‘Effect of FMN on oxidation of glycolic acid

by wheat sap . . . . . . . . . . . . . . .

Effect of light during growth and addition
of FMN to sap upon enzyme activity . . . .

Flavins content in Thatcher wheat leaves .

Flavins recovered from 1.6 x 10"7 11 moles
FAD, after incubation with wheat sap . . .

Relative levels of apoenzyme in green and
etiOlated tissues 0 e e e e e e e e e e e e

Ammonium sulfate precipitated enzyme from
green and etiolated wheat sap . . . . . . .

Q82 of cell-free etiolated sap after 18
hours preincubation . . . . . . . . . . . .

Qg of preincubated dialyzed ammonium
squate precipitate from green wheat . . .

ix

Page

14

26

28

29

3O

32

33

35

37
39

42

44

46

55

XVI.

XVII.

XVIII.

XIX.

XXI.

XXII.

XXIII.

XXIV.

XXV.

XXVI.

XXVII.

XXVIII.

XXIX.

of preincubated dialyzed ammonium
squate precipitate from etiolated
WhCBteeeeeoeeeeeee-eeoee

Effect of spraying on enzyme activity

oratiolatedwheat.............

Requirements of green pea mitochondria
for cytochrome c and effect of glyoxylate

on citrate and succinate oxidation . . . . .

Dependency of P/O ratio upon concentrations
of substrate and phosphate and the duration

of the experiment . . . . . . . . . . . . .

Effect of the sequence of addition of re-
action compcnentson.oxidation of green pea

mitOChondrla...............

Oxidative phosphorylation by pea
mitOChondria O O O O O O O O O O O O O O 0

Effect of glycolate and glyoxylate on
citrate and succinate oxidation by green

peamltOChondrla. 0 scene 0000..

Effect of glycolate and glyoxylate on
citrate and succinate oxidation by
etiolated pea mitochondria . . . . . . . .

Oxidation of oxaloacetate by green pea
mitochondria...............

Effect of glyoxylate and oxaloacetate on
citrate and 11-ketoglutarate oxidation
by green mitochondria . . . . . . . . . .

Effect of thioglycolate on succinate
oxidation by green pea mitochondria . . .

Effect of glyoxylate and FMN on succinate
oxidation by green pea mitochondria . . .

Effect of glyoxylate and glutamate on
succinate oxidation . . . . . . . . . . .

Reversal of glyoxylate inhibition of
succinate oxidation by cysteine . . . . .

Page

56

58

62

67

68

7O

74

80

83

88

91

92

94

96

XXXI.

xi

Page

Effect of glydxylate, tyrosine, 3,4-
dihydroxyphenylalanine (DOPA), and
ascorbic acid an oxidation of
succinate............... 97

Effect of glyoxylate on citrate and
succinate oxidation i3 vivo . . . . . . . . .

1.

7.

8.

9.

10.

11.

12.

15.
14.

FIGURES

Glycolic acid oxidase from green and
etiolated Thatcher wheat leaves . . . . . . . . .

Effect of flavins on enzyme activity of
etiOlated Wheat Bap e e e e e e e e e e e e e o

Preincubation of etiolated cell-free wheat sap .

Preincubation of (NH4)QSO4 precipitate of
green Bap e e e e e e e e o e e e e e e e e e e e

Effect of ATP-glucose-hexokinase system and
yeast coenzyme concentrate on succinate
oxidation by green pea mitochondria . . . . . . .

Substrate inhibition of citrate oxidation . . . .

Effect of phosphate concentration on oxygen
uptake and P/O ratio. Substrate 20l/emoles
Of Citrate o e e e e e e e e e e o e e e e_e e 0

Effect of phosphate concentration on 02 UP-
take and P/O ratio. Substrate, QOl/Lmoles

Of malate e e e e e e e e e e e o e e e e e e e 0

Effect of glyoxylate on citrate and succinate
oxidation by green pea mitochondria . . . . . . .

Effect of glycolate and glyoxylate on malate
oxidation by green pea mitochondria . . . . . . .

Effect of glyoxylate on oxalosuccinate and
cc-ketoglutarate oxidation by green pea
mitOChondria e e e e e e e e e e e e e e 0

Effect of glyoxylate on citrate and succinate
oxidation by etiolated pea mitochondria . . . . .

Single step oxidation of cL-ketoglutarate . . . .
Effect of various glyoxylate concentrations on

oxidation of succinate by green pea mitochondria.

xii

Page

25

41
47

55

61
63

64

65

75

77

78

82
86

89

xiii

Page

15. Inhibition of various levels of succinate
, oxidation by glyoxylate . . . . . . . . . . . . . 100
16. Glyoxylate inhibition of malic dehydrogenase . . 103
17. Glyoxylate inhibition of lactic acid dehydro- . 105

O O O O C O O O

genaee............

18. Non-enzymatic condensation of glyoxylate and
glycine, at pH 10.5, as reproduced from
BeekmanDK-2Charteeeeeeeeeeeeeoo107

19. Non-enzymatic reaction of 0.1 M glycine with
0.1 M glyoxylate at room temperature and pH 8
10

10. C O O O O O O O O O O O O O O O O C O C

20. Effect of glyoxylate and DPN on malate
oxidation by green pea mitochondria . . . . . . . 112

INTRODUCTION

INTRODUCTION

Interest in the metabolism of glycolic acid is a
half-century old and a great number of reports have appeared
concerning this problem.

Glycolic acid, in the presence of the enzyme glycolic
acid oxidase, is metabolized to glyoxylic acid and this, in
turn, to glycine or oxalic acid or carbon dioxide and formic

acid as represented schematically below:

 
 
 

COOH
Glycolic acid I
CHQOH
FMN*
Glycolic acid oxidase
02
COOH
Glyoxylic acid I + H202
CHO
H O
l 2 2 Glycolic acid
Transaminase oxidase, FMN,
002 + H20 202
COOH + COOH
CH2NH2 HCOOH COOH
Glycine Formic acid Oxalic acid

 

* The following abbreviations are used: FMN, ribo-
flavin-S-phosphate; FAD, flavin adenine dinucleotide; DPNH,
diphosphopyridine nucleotide (reduced); DPN, diphospho-
pyridine nucleotide (oxidized); ATP, adenoisine-5'-tri-
phosphate; DOPA, dihydroxyphenylalanine.

"r a...

 

 

Glycolic acid arises as an early product of the
photosynthesis (1, 2) and it is an important metabolically
intermediate (3) of wide distribution in plant tissues (4).
The enzyme glycolic acid oxidase is very widely distri-
buted in plant tissue (4, 5). When combined with glyoxylic
acid reductase, which catalyzes the reverse reaction to con-
vert glyoxylic acid into glycolic acid, the system has been
considered as a possible terminal oxidase (6, 7). Glycolic
acid and glyoxylic acids function as intermediates in the
biosynthesis of glycine and serine. On the other hand,
glyoxylic acid, as the oxidation product of glycolic acid,
inhibits many oxidative reactions, and a number of reports
have been devoted to this problem (82, 86, 88).

The present report concerns the activation of
glycolic acid oxidase and the effect of glycolate and its
oxidation product, glyoxylate, upon the respiration of

plant mitochondria.

LI TERATURE REVIEW

LITERATURE REVIEW

Glycolic acid seems to be present in microorganisms,
microflora, plants, and animals. Also, it has been reported

to be in the organic matter of unmanured soil (8).

Glycolic Acid in Microorganisms

In 1932 it was reported that calcium and sodium ace-
tates were converted to glycolic and glyoxylic acids by
Aspergillus ni er, and that glycolic acid was converted
rapidly to glyoxylic acid (9). Glycolic acid as an inter-
mediate product in the oxidation of acetate by E, 29;; also
has been reported (10). A mutant strain of Paeudgmgnae
eagghgrgphila, capable of adapting D-arabinose as its sub-
strate, produces glycolic acid (11). D—arabinose is oxidized
to D-arabono- g-lactone, the lactone is hydrolyzed to D-
arabonic acid which is dehydrated to 2-keto-3-deoxy-D-
arabonic acid and finally this acid is hydrogenated and
cleaved to yield pyruvic and glycolic acids. The carboxyl
group of pyruvic acid is derived from C1 of arabinose, while
the carboxyl group of glycolic acid arises from C4. Cell-
free extracts of Penicillium Chrysogenum are able to oxidize
glycolate to glyoxylate (12). This oxidation is stimulated
by the addition of flavin and pyridine nucleotide cofactors.
In crude extracts the glyoxylate disappears rapidly and the

only detectable compound produced from glycolate-C14

metabolism is glycine. In the biosynthesis of glycine by
Neurospoga mutant, glycolic acid is oxidized to glyoxylic
acid which, in turn, is transaminated to glycine (13).
Cell-free extracts of Pseudomonad synthesize glycine by
transamination of alanine, aspartic acid and glutamic acid
with glyoxylic acid (14). The non-enzymatic transamination
with glyoxylic acid and various amino acids has been reported
also (15). Glycine may be converted in the reverse reaction
to glyoxylate. Washed cells of Pseudomonas aeruginosa, in
the presence of oc-ketoglutarate, ATP and pyridoxal,
metabolize glycine readily to glyoxylic acid (16). It was
suggested that glycine is converted into glyoxylic acid in a
transamination reaction. Another source of glyoxylate in
microorganisms is the cleavage of isocitrate to succinate
and glyoxylate (18, 19, 20). Glyoxylic acid may enter the
tricarboxylic acid cycle in microbial metabolism by the
condensation with acetate to form malate (17, 18, 19).
Cell-free extracts oflg. gel; convert anaerobically glyoxylic
acid to hydroxypyruvate (21, 108). In this process, one
mole of 002 accumulated per two moles of glyoxylate added
and tartronic semialdehyde was identified as the first pro-

duct which, in turn, was metabolized to hydroxypyruvate.

 

Glycolic Acid in Animal Tissues

It has been reported that as early as 1919, human

tissues were capable of converting glycolic acid into

glyoxylic acid, formaldehyde, and formic acid (22). The
oxidative decarboxylation reaction of glyoxylate to formats
and carbon dioxide has been demonstrated with rat liver
mitochondria (23). Perhaps the first report concerning
glycolic acid as a precursor of glycine in intact animals
(rabbit) appeared in 1941 (24). Glycolic acid, when ad-
ministered with benzoic acid, caused an increase in the
rate of excretion of hippuric acid. This increase in the
rate of excretion of hippuric acid was interpreted as
evidence of the ability of the rabbitt to convert glycolic
acid to glycine. Employing isotopic procedure for the study
of glycolic acid metabolism in the intact rat, it was found
that this acid was converted to formic acid, and that the
carbon of formic acid arose from ci-carbon of glycolic acid
(25, 26). Similar results were obtained with cz-labeled
glycine or x-labeled glyoxylate. Glycolic and glyoxylic
acids, when injected intraperitoneally into rats, are con-
verted rapidly into glycine (27); whereas, acetate, formats,
or oxalate are not. Oxalate was metabolically inert and
upon addition of glyoxylate, an accumulation of oxalate was
observed in the rat. When rat-liver homOgenate is incubated
with glycolate or glyoxylate, there was found a trifold in-
crease of glycine content as compared to the endogenous
amount of it. Upon the addition of glutamine to the incu-

bation mixture, the content of glycine formed greatly

increases (28). This indicates not only the capability of
animal liver homogenates to oxidize glyoxylate, but also the
formation of glycine by a transamination process with
glutamic acid. Rat-liver specimens are capable of forming
glycine from glyoxylic acid as a result of transamination
reaction with aspartic acid, asparagine, glutamic acid, and
glutamine, both aerobically and anaerobically (29). When
glycine was incubated with bovine spermatozoa, glyoxylic
acid, formic acid, and carbon dioxide was formed (30). This
indicates that glycine was metabolized and that the pathways
involved may be similar to those observed in other mammalian

tissues.

Glycolic Agid in Plants

9 When glycolic acid was added to freshly ground
barley leaves, there was observed an increased oxygen uptake
and an accumulation of glyoxylic acid (31). Glycolic acid
is oxidized more rapidly by chloroplast preparations in the
light than in the dark. It was suggested that this
oxidation is associated with some function of chlorOplast in
the light (32).

It was observed that oxidation of chlorophyll
paralleled the oxidation of glycolic acid by leave homo-
genates (33, 34). The rate of chlorophyll disappearance was
proportional to the concentration of glycolate added to the

barley sap (35). It has been assumed that the active

oxidizing agent was the peroxide of glyoxylic acid, but it
is more likely that the H202 produced oxidized the chloro-
phyll. If the accumulation of peroxides was prevented by
the addition of ascorbic acid or'phenols, the chlorophyll
was not oxidized. On the other hand, in the absence of a
carbonyl compound, considerable peroxides could accumulate
without the chlorophyll undergoing oxidation (36). The
oxidation of chlorophyll takes place in the simultaneous

presence of glycolic acid and nitrites (37). Since the

oxidation of glycolic acid takes place without nitrites,

then this process precedes the oxidation of chlorophyll. In
either the light or the dark the amount of nitrates declines
when glycolic acid is detectable in the system and the re-
sulting nitrites are rapidly converted into hydroxylamine
and other substances in a reaction in which glycolic acid
participates.

When detached tobacco leaves were vacuum infiltrated
with the solution of glycolic acid, labeling was found in
oxalic, succinic, malic, citric, and acetic acids, both in
the light and in the dark (38). In the light, malic acid
had a specific activity almost four times higher than citric
acid; whereas, in the dark, citric acid had a specific
activity four times higher than malic acid, although it had
a lower total content of labeled carbon. On the basis of

this observation, an assumption was made that glycolic acid

can participate also in a tricarboxylic acid cycle. This
assumption is supported by the finding that over twice as
much labeled carbon from glycolic acid accumulated in
glutamic acid in the dark as in the light (39), and by the
presumption that a tricarboxylic acid cycle, active in the
dark, may be suppressed by light.

Carbons 1 and 2.0f ribose are known to be precursors
of the at-, and carboxyl carbons of glycine, respectively
(40, 41), and glycine, in turn, arises from glycolic acid
(42, 43). Therefore, glycolic acid should be synthesized
ig_yiyg from carbohydrates by a transketolase of trans-
aldolase type of reaction. Recent work, in fact, has shown
that glycolic acid is produced from a transketolase attack
on fructose-6-phosphate in the presence of excess ferricyanide.
When tobacco leaves were infiltrated with ribose-1-C‘4, the
radioactivity appeared in oc-carbon of both glycolate and

glycine (33).

During the steady-state C14

02 photosynthetic experi-
ments with Chlorella plrenoidosa, much glycolic acid was
excreted into the medium (45). This phenomenon was

specific for glycolic acid, as it was the only product ex-
creted. This secretion of glycolic acid into a medium was
extended to whole chloroplasts during the photosynthesis
(46). These results, concerning the formation of glycolate

during the photosynthesis, were confirmed on a quantitative

basis with Chlorella (47). From two molecules of carbon
dioxide fixed there was found one molecule of glycolic acid.
Parallel with this confirmation of glycolate formation
during the photosynthesis, another group reported that
during photosynthesis by Chlorella, carbon dioxide was con-
verted directly into glycolic acid (48). The only postulated
intermediate was a radical, probably (CHO0), which was be-
lieved detectable by electron spin resonance. This latter
group claimed that they had found a second photosynthetic
pathway; however, their results have not been verified.

Glycolic acid is metabolically very active in plants.
In tobacco leaf homogenates, glycolate is readily converted
into glyoxylate, carbon dioxide, and formats (49). Formic
acid is incorporated directly into the~AQ-carbon of serine
and into choline (50). The major two products of glycolate
metabolism by plants are glycine and serine (42, 43). The
carbon atoms of the glycolic acid become the corresponding
ones in glycine. The carboxyl carbon of serine arises from
carboxyl carbon of glycolic acid and both the ar-and fl -
carbons of serine come from the cz-carbon of glycolic acid.
Serine synthesis in etiolated plants exposed to the light
develops parallel with activation of glycolic acid oxidase
and this indicates that the major pathway for serine synthesis
during the photosynthesis is via the glycolic acid pathway
(51).

10

By a transamination reaction, glycine arises from
glyoxylate which is an oxidation product of glycolate (52,
53, 54). Glyoxylic acid may also enter the citric acid
cycle by a reaction catalyzed by malate synthetase and
participate in synthesis and energy production in plant
material. Malate synthetase, together with other enzymes of
the glyoxylate cycle, were first demonstrated in plants of
caster beans (55). Operation of glyoxylate cycle in plant
metabolism was confirmed for germinating peanuts and castor
beans (56) and also for excised cotyledons from etiolated
peanut and sunflower seedlings (57). Occurrence of malate
synthetase is not limited to the oily, germinating seeds or
young seedlings of these seeds. Several other non-fatty
tissues, such as, five-day old pea cotyledons, showed also a
high malate synthetase activity. However, in mature leaves
this enzyme has been detected only in trace amounts (58),
and cannot account for glyoxylate metabolism in tissues of
leaves.

Since glycolate and glycine are interconvertible in
plant metabolism, the o<-carbon of glycolic acid may be
utilized for methyl group synthesis. This was shown to be
the case in the experiments with tobacco plants, where the
o<-carbon of glycolate or glycine was incorporated into
N-methyl group of nicotine (59, 60) and the a1-carbon of

glycolate into O-methyl groups of lignin (60). The at -carbon

11

of glycine was incorporated into pectinic acid in radish
plant metabolism, and 70% to 80% of the radio-activity of

pectinic acid was located in the methyl ester carbon (61).

Glycolic Acid Oxidase
Glycolic acid oxidase catalyzes the oxidation of

glycolic acid to glyoxylic acid. The first report concern-
ing the existence of such an enzyme in animal tissue appeared
in 1940 (63). Glycolic acid oxidase was found to be widely
distributed in green plants (5) and it was described in some
detail (64). In the latter publication it was reported that
the enzyme appeared to be specific for L-aL-hydroxymono-
carboxylic acids; that is, glycolic, lactic, and a(-hydroxy-
n-butyric acid. Glycolic acid oxidase was isolated in
crystalline form from spinach leaves (65).

The prosthetic group of the enzyme is riboflavin-5-
phosphate (66, 67, 68, 69, 72). A.Michaelis' constant
2.4 x 10‘3 (64) or 3.8 x 10"4 (69) has been reported which
indicates a high affinity of the enzyme for the substrate.
The enzyme catalyzes the oxidation with molecular oxygen of
glycolic acid to glyoxylic acid, and of lactic acid to
pyruvic acid in both plants (37. 49) and animals (70, 71).
In cell-free plant homogenates, a byproduct of this oxi-
dation, hydrogen peroxide, non-enzymatically oxidizes
glyoxylic acid to carbon dioxide and formic acid (49, 67,

68). However, the enzyme glyoxylic acid oxidase has been

12

reported in rat liver tissue which is responsible for this
oxidative decarboxylation (23). In the presence of excess
catalase, the hydrogen peroxide byproduct is destroyed and
the glyoxylic acid is not oxidized to formic acid and 002
but accumulates (67, 72). Molecular oxygen liberated from
the catalase attack on H202 may be used to oxidize another
portion of glycolate. Glyoxylic acid in plants is partially
oxidized to oxalic acid. In litre this oxidation is catalyzed
by the same glycolic acid oxidase and riboflavin-S-phosphate
that oxidizes glycolic acid (73, 100). The nearly ir-
reversible reaction of reduction of glyoxylate to glycolate
is catalyzed by glyoxylic acid reductase at the presence of
DPNH (74). This enzyme has been isolated from tobacco
leaves (75) and reduces glyoxylate to glycolate or hydroxy-
pyruvate to D-glycerate. Recently Zelitch has discovered
another glyoxylic acid reductase which uses TPNH rather than
DPNH. The role of these two reductases in plant tissue is
not known (62).

Glycolic acid oxidase seems to be absent in roots
(49. 76), although roots contain small amounts of both
glycolic and glyoxylic acids (77). When exposed to the
light for a sufficient length of time to develop a green
color, root tissue shows glycolic acid oxidase enzymatic
activity (76).

The activation of glycolic acid oxidase is of unusual

13

and particular interest. From the published data we know
the activity of the enzyme in plant material, expressed as
Qgé, varies as shown in Table I. Glycolic acid oxidase is
active as isolated from green leaves (5, 64, 69). On the
basis of Qg2’ it is much less active (about one-tenth) as
isolated from etiolated leaves (5). However, etiolated

leaves are able to convert glycolic-C14

acid to glycine
(43). The activity of the enzyme can be increased greatly
iggyiyg_by exposure of plants to the light for a few hours
(5, 34, 78). The activity of the enzyme can also be in-
creased ig,yiyg in the dark by feeding the leaves glyco-
late (5, 78). From the cell-free homogenate of etiolated

leaves, the activity of the enzyme can also be increased ig

vitro by incubation with glycolate for several hours (5).

Role of Glycolic and Glyoxylic Acid in Plant Respiration

As mentioned in the introduction, there has long
been an interest in glycolic acid and glyoxylic acid
oxidation and reduction as a possible terminal oxidase
system. Zelitch (79) has published data which he interpreted
as indicating that a substantial part of the plant respir-
ation could be accounted for by this system. Yet, last
year Zelitch (80) showed that glycolic acid oxidation by
plant mitochondria produced no energy, for the P/O ratio
was zero. These two facts conflict because a major purpose

of respiration is the production of energy.

14

TABLE I

Activity of glycolic acid oxidase

from plant tissues (5)

 

 

N
002
ޤource of enzygp
Sap of green leaves 50 to 200
Sap of etiolated leaves 5 to 10
Sap of etiolated leaves which
were sprayed with glycolate
24 hours before harvest 100 to 300

Etiolated leaves sap, which
was incubated with glyco-
late for 18 hours at 0°C. 50 to 100

15

Beevers and Kornberg (55) have shown that glyoxylic
acid is a major metabolic constituent of the glyoxylic acid
cycle in germinating plant tissue. However, they were not
able to demonstrate an active glyoxylic acid cycle in older
plant tissue. Therefore, this function seems unlikely in
plants during most of their growth.

The main pathway of respiration in higher plants in~
eludes thetricarboxylic acid cycle and proceeded by the
initial breakdown of carbohydrates and fats to pyruvate and
acetyl-CoA, which are then completely oxidized in the citric
acid cycle.

Glyoxylic acid is known to inhibit the oxygen uptake
of various respiring tissue suspensions. Perhaps the first
report concerning this phenomenon appeared in 1943 (81), and
this was observed on animal tissues (liver, kidney) where
glyoxylic acid exhibited strong inhibitory effects on
tissue respiration.

In the course of study of metabolism of phosphorous
deficient plants, both citric acid and glyoxylic acid were
found to accumulate in plant tissues (82). The accumulation
of citric acid in phosphorous deficient plants was ascribed
to the enormous formation of glyoxylic acid in this plant
followed by the inhibition of citric acid oxidation by
glyoxylate. When glyoxylate was incubated with oxalo-

acetate in rat liver homogenates, it produced a severe

16

inhibition of citrate oxidation (83, 84).

The inhibitory effect of glyoxylate was also ob-
served on succinate respiration by plant mitochondria
preparations (85). In spite of the inhibition of the total
oxidation of succinate or citrate by glycolate and glyoxy-
late in spinach mitochondria, no lowering of the P/O ratio
for either of these substrates was observed (80).

The addition of glyoxylate to rat liver homogenates
produced an inhibition of the oxidation for all tricarboxylic
acid cycle intermediates (86). Simultaneously, with this
inhibition in all cases, some increase in the accumulation
of citrate was observed. The highest inhibition by
glyoxylate and a large accumulation of citrate was observed
when oxaloacetate was the substrate. This maximal inhibition
of oxidation by glyoxylate when oxaloacetate was used as the
substrate and a concurrent, enormous accumulation of citrate
was ascribed to the reaction between glyoxylate and oxalo-
acetate and formation of an inhibitor of citrate metabolism.
This inhibition was thought to be oxalomalic acid, which
would arise from the condensation of glyoxylate and oxalo-
acetate.

In a study of the effect of o<-ket0glutarate and
aspartate on succinoxidase from human mitochondria, it has
been found that neither of these acids had a direct in-

hibitory effect on succinate oxidation (87). The actual

17

inhibitor was oxaloacetate produced by transamination when
both aspartate and oL-ketoglutarate were present in the
incubation of succinate with mitochondria preparation.

When purified aconitase from pig heart was incubated
with sis-aconitate, it has been observed that 100% in-
hibition of aconitase activity upon addition of both
glyoxylate and oxaloacetate to the incubation mixture (88),
and the hypothetical oxalomalic acid was assumed to be
responsible for this inhibition of aconitase. The chemical
nature of this compound is reported to be under investi-
gation, but, as yet, no further report on it has appeared.

Since glycolic and glyoxylic acids are easily inter-

convertible in plants, inhibitory effect of tricarboxylic

acid cycle operation could be exhibited by either one,

glycolate or glyoxylate, when tested i3 vivo (80).

MATERIALS AND METHODS

MATERIALS AND METHODS

Pleat Material

Thatcher wheat, Alaska peas, and Sacramento barley
were grown in sand with Heagland solution (89). If green

material was needed for the study, the plants were grown in
a greenhouse; in order to obtain etiolated plant tissue,
the plants were grown in a totally dark room. For experi-
ments with effect of light on glycolic acid oxidase activity
during the growth, Thatcher wheat plants were grown in the
dark room and before harvest they were exposed to the day-

light of "September intensity" (2000-3000 foot candles) for

a desired length of time. The age of the plants used for

the study was eight to ten days. Swiss chard leaves were

obtained from the plants grown under field conditions.

Preperation of Tissue and Enzyme

In order to obtain sap from the plant material for

the study, harvested leaves were ground in a cold mortar

with some white sand. The sap was squeezed through a double-

layer of cheesecloth and the pH of the homogenate was
quickly adjusted to about pH 8 with potassium hydroxide, using

a Beckman pH meter, Model G. After centrifugation for five

minutes at 200 x G in an International Portable Refrigerated

Centrifuge, Model PRr2, sediment was discarded and the pH of

19

the cell-free sap was adjusted to 8.3 when glycolate was

used as a substrate, or to pH 7.3 when glyoxylate was used

as a substrate. During the preparation of the enzyme ex-

tract, the temperature of the sap was maintained between 0

and 2°C.
For preparation of whole chloroplast, cytoplasmic,

and protoplasmic fractions of the plant cell, the procedure

by Arnon, et a1 (90), was followed. Throughout this pro-

cedure the pH was maintained at 8.3, which is the optimum

of glycolic acid oxidase.

Mitochondria-free supernatant was obtained by sedi-
menting the precipitate at 75,000 x G for one hour in a

Spinco Ultracentrifuge,, Model L.
An ammonium sulfate precipitate of glycolic acid

oxidase was prepared from the cell-free plant sap. A 140

gm. per liter of ammonium sulfate was added to the sap at

pH 5.3 and an inactive protein was removed by centrifugation

at 4500 x G and discarded (65). The glycolic acid oxidase
protein fraction was sedimented by an additional 80 gm. per

liter of ammonium sulfate, stirred into the supernatant.

After centrifugation at 4500 x G for 90 minutes, the super-

natant was discarded, and the precipitate was taken up in

0.1 M phosphate buffer at pH 8.3 and dialyzed against cold

water for 4 to 6 hours.

Mitochondria from plant tissues were isolated

20

essentially after the procedure of Ohmura (91), using one

volume of leaves and two volumes of grinding medium of the

following composition: 0.45 M sucrose, 0.05 M manhitol,

0.05 M boric acid, 0.03 M potassium citrate, 0.05 M tris

(hydroxymethyl) aminomethane chloride (Tris), and 0.01 sodium

ethylenediamine tetraacetate (versenate). The pH of the

grinding medium was adjusted to 7.6 and this, when used in

two volumes with both green or etiolated pea leaves, gave

the desired pH 7.2 in the homogenate. Plant debris, chloro-

plasts, whole cells, et cetera, were removed by centrifuging

for seven minutes at 600 x G at 0°C. The supernatant was

next centrifuged at 10,000 x G for 20 minutes and the resi-

due of particles was homogenized again with a desired volume

of the washing medium. The washing medium was of the follow-

0.3 M sucrose, 0.05 M tris (hydroxymethyl)
The

ing composition:

aminomethane chloride (Tris) and 2 x 10"4 M versenate.

pH of the washing medium was adjusted to 7.5.

Assays4agd Analyses
The oxygen uptake of both cell-free sap and mito-

chondria preparations was measured by manometric techniques
in 23 ml. Warburg vessels with an atmosphere of air, shaken
at 120 oscillations per minute. Temperature was maintained
at 30°C. All assays for glycolic acid oxidase activity in

plant sap were in 0.033 M final concentration of phosphate

21

buffer. Each flask contained one ml. of sap. The final
volume of reactants was 3 ml. and the pH was 8.3.

The standard reaction components for experiments with
isolated mitochondria were (80): sucrose, 3004a¢moles;
MgSO4, 10 /zmoles; potassium phosphate buffer at pH 7.0,
37’Jcmoles; yeast coenzyme concentrate, 1 mg; ATP, 2/Azmoles;
mitochondria from 1.5 g. plant tissue and substrate as will
be specified. If deviation from this standard assay pro-
cedure was made, this will be specified with the corresponding
graphs or tables. The reaction mixture was combined at zero
time with glucose, 50 fiomoles, and yeast hexokinase, 0.2 mg.
which had been placed in the side arm. The final volume of
reactants was 3.9 ml. and 0.2 ml. of 203 KOH were placed in
the center well. Reaction was usually followed for one hour.

For nitrogen determination, the micro Kjeldahl
method was employed (92). The /;l 02 taken up per mg.
tissue nitrogen per hour is designated as Qg2.

Phosphorous was determined colorimetrically by the
Fiske-Subbarow method (93). After completion of manometric
measurements in the Warburg apparatus, the reaction mixture
was deproteinized with 0.5 ml. of 20% trichloroacetic acid,
the precipitate was removed by centrifugation and the ortho-
phosphate remaining in the supernatant fluid was determined.

The amount of orthophosphate esterified was determined by

the difference between the contents of orthophosphate in the

22

endogenous reaction mixture and in the appropriate substrate
reaction. For this determination a Coleman Junior Spectro-
photometer, Model 6A, was used.

The flavin content was determined fluorometrically
(94, 95) by using an Electronic Fluorometer, Model 12B, of
Coleman Instruments, Inc.

Oxaloacetic acid was determined by the oxidative de-
carboxylation method (96). Malic and lactic dehydrogenase
activity was measured spectrophotometrically (97) in the
Beckman Spectrophotometer, Model DU. If malate was used as
a substrate, its oxidation with DPN in the presence of malic
dehydrogenase was followed by the measurement of the DPNH
produced at 340 mp.

Lactic dehydrogenase activity was also measured in
the Beckman Spectrophotometer in a similar way, that is, by
following lactate utilization by the enzyme as reflected in
the optical density changes for the presence of DPN.

For the recording of absorption spectra, a Beckman,

Model DK-2, instrument was used.

Source of Chemicals

From the Nutritional Biochemical Corporation, Cleve;
land, Ohio, was obtained riboflavin-S-phosphate (FMN),
flavin adenine dinucleotide (FAD), diphosphopyridine
nucleotide reduced (DPNH), L-glutamic acid, L-cysteine

 

23

hydrochloride, L-tyrosine, dihydroxyphenylalanine (DOPA),
glycine, acetyl phosphate (diAg), sucrose, D-mannitol, and
the following organic acids: glyoxylic (sodium salt), citric,
cis-aconitic, isocitric (trisodium salt), oxalosuccinic
(barium salt 60%), aL-ketoglutaric, fumaric, L-malic

(active), oxaloacetic, malonic, and ascorbic acid.

Sigma Chemical Company, St. Louis, Missouri, was the
source of: Adenosine-B'-triphosphate (ATP)-disodium salt,
yeast hexokinase (type II), yeast coenzyme concentrate
(Stock No. 202-30), malic dehydrogenase (Stock No. 410-9),
cytochrome c (type II, from horse heart), and tris
(hydroxymethyl) aminomethane chloride (Tris).

From Eastman Organic Chemicals, Rochester, New Yerk,
was obtained glycolic acid (calcium salt), D-glucose,
succinic acid, lactic acid (85%). 1-amino-2-naphthol-4-
sulfonic acid and trichloroacetic acid (practical).

Diphosphopyridine nucleotide (DPN) was obtained from
Pabst Laboratories, Milwaukee, Wisconsin.

Riboflavin was from Merck and Company, Rahway, New
Jersey; potassium citrate, Mallinckrodt Chemical Wbrks, New
York; boric acid, Baker Chemical Company, Phillipsburg, New
Jersey; and sodium ethylenediamine tetra-acetate (Versenate)

was obtained from Matheson Company, Norwood, Ohio.

 

PESULTS AND DISCUSSION

RESULTS mm DISCUSSION.
A. GLYCOLIC ACID OXIDASE IN PLANTS

Organ and Cellular Distribution of_§lycolic Acid_0xidase

,A relative comparison of the glycolic acid oxidase
activity as isolated in the sap from green and etiolated
leaves of Thatcher wheat is shown in Figure 1. This con-
firms previous reports of this phenomenon (5, 34, 69).

Most of the work published on glycolic acid oxidase
in plant tissue concerns the enzyme as isolated from green
leaves and shoots. No enzyme can be extracted from root
tissue unless the roots were exposed to the light and until
they developed a green color (76). As will be discussed
later, the enzyme activity is greatly stimulated from sap
of etiolated shoots by both light and by the addition of
FMN. Therefore, the effect of FMN upon activation of the
enzyme in sap from roots was also studied (Table II).
Shoots of green pea seedlings showed high glycolic acid
oxidase activity without added FMN, but roots of the same
plants showed no enzymatic activity regardless of the
addition of FMN. Presumably the roots contain no enzyme
or apoenzyme for this oxidation.

Glycolic acid oxidase has been reported as being

absent in the sheaths of gramineous plants (98), although

25

FIGURE 1
Glycolic acid oxidase from green and

etiolated Thatcher wheat leaves

 

 

 

 

 

 

 

240 5-,-
i
200t- ///O
l//l
160- .///
120..
e
.M
‘3 , ///*v***- Green
a l ,
:1 A /
c9’ ,
F‘ 80'- (3»
3‘, ,.
40 F (j/
,fi{//’ ‘r-Etiolated
!,/ . .q
i/ _i.l1 .L #‘@i ‘t ’— 4 I
70 20 3o 40 50 60

Time in Minutes

Each Warburg flask contained

50 linoles of glycolate.

TABLE II

26

Glycolic acid oxidase from shoots and

roots of green pea seedlings

 

F."

Sap and substrate

Time

!

 

10 min.‘ 25 min.

45 min. 60 min.

 

 

 

Shoot sap:

IO/Lmoles glycolate

IO/Lmolgs glycolate+
1 x 10' M FMN
Root sap:

10,“.1110188 glycolate

10,u.molfis glycolate-+-
1 x 10' M FMN

 

[sol 02 uptake/ml. sap/hr.

 

 

64

75

 

154 158 158
172 169 181
2 3 5

 

 

 

 

27

adjacent green leaves showed normal enzymatic activity. Sap
from the sheaths and leaves of both green and etiolated
wheat seedlings were prepared after careful visual sepa-
ration of the sheaths. In the sheaths of green plants about
4 per cent as much enzyme activity on a Q82 basis was found
as in the leaves of the same plants (Table III). Upon
addition of FMN to the saps of both leaves and sheaths, the
enzymatic activity doubled for the green leaves but increased
about 39 fold for the green sheaths. As a result both
tissues of green seedlings had nearly equal enzyme activity
on a Q82 basis. The data indicate that a large amount of
apoenzyme was present in green sheaths, and in order to re-
veal it, the addition of coenzyme was needed. These results
alter the previous concept that the enzyme was not present
in the sheaths (98). Both etiolated tissues, leaves, and
sheaths showed very low glycolic acid oxidase activity with-
out the addition of FMN. When FMN was added, the enzymatic
activity was greatly increased in both tissues, but the
enzymatic activity of etiolated sheaths was only about one-
third as great as that of the leaves.

Distribution of the enzyme in isolated cellular
fractions from Swiss chard is shown in Table IV and from
Thatcher wheat in Table V. Both the chloroplasts from
green tissue (Table IV), and mitochondrial particles sedi-

mented between 2,000 and 75,000 x G from etiolated tissue

28

TABLE III

Glycolic acid oxidase in leaves and sheaths

of Thatcher wheat

 

 

 

 

 

 

FMN final Q32
Kind of tissue concentration
0 55.0
Green leaves 1 x 10'4 M 107.6
0 2.2
xSheaths from green
plants 1 x 10“4 M 89.4
0 6.6
Etiolated leaves 1 x 10'4 M 55-2
0 5.8
Sheaths from etiolated l 4
plants 1 x 10‘ M 16.4

 

 

 

Each flask contained 20/Lmoles of glycolate.

 

29

TABLE IV

Location of glycolic acid oxidase in fractions

from Swiss chard

 

 

Cell constituent Q82

Chloroplast 52.4

Protoplasm 345.4
1 Cytoplasm 338.0

 

 

 

 

Each flask contained SO/gtmoles of glycolate.

Location of glycolic acid oxidase in fractions

Substrate

TABLE V

from etiolated Thatcher wheat

Cell Constituent*

L

30

 

Supernatant J Mitochondria

 

 

1 x 10‘1+ M FMN
LIto/«moles glycolate
FMN + glycolate

I'

Imal 02 uptake/hour

 

4
1 6
139

 

 

14
9
36

 

* After 1 hour centrifugation at 75,000 x G, sedimented

particles were resuspended in a volume of phosphate

buffer equal to the volume of the supernatant.

 

31

(Table V), showed lower enzymatic activity on a Qgé basis
than the cytoplasmic soluble fraction. These data could be
accounted for by absorption of the enzyme on the surface of
the chloroplasts or mitochondria. Such a phenomenon of ab-
sorption of soluble enzymes is frequently reported (80, 87).
On the other hand, much enzyme activity could have been with
the particles in Ella and was leached out during the iso-
lation procedure. From such data it is generally considered
that the glycolic acid oxidase is predominantly a soluble
cytoplasmic enzyme and not associated with chlorOplasts as
was earlier reported (99). These results confirm an earlier

report concerning the distribution of the enzyme (66).

Activation of theMme by Addflxcess of FMN

Enzyme activation by the addition of FMN to the cell-
free saps is presented in Table VI for wheat and in Table
VII for barley. In order to obtain the maximal enzymatic
activity in both green and etiolated cell-free homogenates
or saps of plant leaves, the addition of FMN was necessary.
The apoenzyme could be nearly completely activated by a
final FMN concentration of 1 x 10'5 M or greater. The maxi-
mum enzyme activity for both green and etiolated leaf ex-
tracts was obtained between 1 x 10"4 and 1 x 10"3 M FMN,
while a 1 x 10‘2 M final FMN concentration was slightly

inhibitory. The maximum increase in enzyme activity upon

32

TABLE VI

FMN activation of glycolic acid oxidase

from green and etiolated plants

 

 

 

 

FMN final
concentration Green sap Etiolated sap
Molar 032 A Q32
0 39.9 4.1
1 x 10-7 38.7 6.9
1 x 10'6 54.5 11.7
1 x 10‘5 95.0 30.4
1 x 10"4 105.0 42.1
1 x 10-3 106.9 40.7
1 x 10'? 95.0 36.5

 

 

 

 

Each Warburg flask contained 20 ;amoles of glycolate.

0%? for FMN, without glycolate, was 2.1 for green sap,
and 1.9 for etiolated sap.

33

TABLE VII

Oxidation of glycolate and glyoxylate
by etiolated barley sap

 

 

 

 

 

Flask additions fiLl 02 uptake/hour
FMN 0
Glycolate 7
Glycolate + FMN 1 150
Glyoxylate 5
Glyoxylate + FMN 42

 

Glycolate or glyoxylate 10 memoles/flask. FMN
was at 1 x 10'4 M final concentration. Final
pH for flasks with glycolate, 8.3, and with

glyoxylate, 7.3.

34

the addition of FMN was higher in etiolated tissue, reaching
values of about 10 fold; whereas, in green material the maxi-
mum increase in enzymatic activity was less than 2.5 fold.

0n the other hand, etiolated plant material always showed, on
the basis of egg, two to three times less enzymatic activity
than green tissue. From either tissue the maximum activity
was never obtained, unless excess FMN was added.

In Table VIII are data to demonstrate the stimu-
lation by FMN of the oxidation of glyoxylic acid by wheat
sap. It has generally been observed that the oxidation of
glyoxylate in the absence of added FMN is extremely slow by
cell-free homogenates or mitochondria preparations from both
green and etiolated tissues. If glycolic acid was the start-
ing substrate and excess of catalase was omitted, the
glyoxylic acid product would be non-enzymatically oxidized
by the hydrogen peroxide formed in the reaction to formic
acid and carbon dioxide (49, 67, 68). In the present
experiments glyoxylic acid was used as the substrate and
FMN was added in varying concentrations. A.many-fold in-
crease occurred in the oxidation rate of glyoxylate by both
etiolated and green tissues. Richardson and Tolbert (100)
have shown that glycolic acid oxidase can oxidize glyoxylate
as well as glycolate, but that the Km for the oxidation of

glyoxylate is much less than for oxidation of glycolate.

TABLE‘VIII

35

Effect of FMN on oxidation of glyoxylic

acid by wheat sap

 

 

 

FMN final Etiolated

concentration Green sap sap
Molar Q32 Qgé

0 2.5 0.0

{1 x 10'7 1.8 0.8
1 x 10‘6 2.4 1.0
1 x 10"5 6.4 1.0
1 x 10'4 27.0 3.4
1 x 10'3 26.7 6.9
1 x 10‘? 30.0 5.4

 

 

 

 

‘—

Each flask contained 20 females of glyoxylate.

There was no effect of FMN alone, without

glyoxylate.

36

Effect of Light plus FMN on Enzyme activity
In order to determine the effect of the length of

the light period and the addition of FMN'to the cell-free
sap on enzyme activity, a series of different experiments
were run with wheat plants all of which were 10 days old
from time of planting the seed (Table IX). The variable was
the duration in hours of exposure to daylight before harvest-
ing of plants. The germinating seedlings appeared above the
surface of the sand after five days and, therefore, the
longest possible exposure of plants to the light was about
four days. The measurable activity of the enzyme without
addition of EMN in cell-free homogenates from Thatcher wheat
leaves which had been grown in total darkness for 10 days
was low (Qgé = 5.3). This activity was stimulated 9.0 fold
by the addition of FMN. These data suggest that the apo-
enzyme was present in the dark-grown plants, but that it was
not active, perhaps due to insufficient FMN. Exposure of
the plants to the light also caused the formation of more
active enzyme and of more apoenzyme.

The complexity of these multiple changes is diffi-
cult to interpret. However, the ratio of Q82 activity in
the presence of added FMN to that without added FMN dropped
from 9 for etiolated tissue to 5 or 6 for any tissue exposed
to light. Saying this another way, there was proportionally

more apoenzyme to holoenzyme in etiolated tissue than in

37

TABLE IX

Effect of light during growth and addition

of FMN to sap upon enzyme activity

 

 

Ratio of:

Exposure Glycolate Glycolate + FMN
t2 light Glycolate + FMN Glzcolate

932 03‘,

0 5.3 47.5 9.0
3 hours 9.8 50.6 5.2
6 hours 10.9 70.2 6.4
12 hours 14.0 92.7 6.6
2 days 19.2 121.8 6.4
3 days 24.6 133.2 5.4
4 days 29.6 158.1 5.3

Each flask contained 10 fcmoles of glycolate. FMN final
concentration was 1 x 10'4 M. Age of all plants at

harvest was 10 days.

38

tissues exposed to light. However, exposure to light also
stimulated the formation of both holoenzyme and apoenzyme
which remained, though, in a.proportion of five to six times
more apoenzyme than holoenzyme. The rapid change in this
ratio from 9.0 to 5.2 after only three hours in light oc-
curred before significant new protein synthesis or growth,
as evident by the fact that the total enzyme activity in the
presence of FMN was still the same in both cases. This sug-
gests that exposure of plants to light had two effects upon
glycolic acid oxidase activity. Light immediately was
responsible for an increase in holoenzyme from a reservoir
of apoenzyme. This would not be caused by protein synthesis,
but by an effect upon the coenzyme. Light also stimulated
the slow synthesis of new protein or enzyme so that at the
end of two to four days the green plants contained much more

enzyme than plants of comparable age kept in the dark.

Flayin Content of Wheat_Leaves

The total flavin content of Thatcher wheat leaves

 

does not differ significantly between green or etiolated
tissue (Table X), but the distribution of the flavins among
FAD, FMN, and riboflavin was strikingly different in these
two types of leaves. FAD content of etiolated leases was
twice as high as that in green leaves. 0n the other hand,

the FMN content was two times greater in green than in

TABLE X

39

Flavins content in Thatcher wheat leaves

 

6/g Dry sample

Calculated molarity*

 

 

 

 

Flavin
Green Etiolated Green Etiolated
AD 46 108 4.1 x 10-9 6.0 x 10'9
93 53 1.3 x 10"8 4.5 x 10-9
Riboflavin 2 5 4.2 x 10-‘0 8.9 x 10-10
Total 141 166 -- --

 

 

 

 

 

 

* Calculated on the basis of 3

of flavin per gram of

dry sample and amount of sap obtained from an

equivalent quantity of fresh tissue.

 

4o

etiolated tissue. These results are consistent with the
fact that green tissue has much more of the active FMN re-
quiring glycolic acid oxidase. However, the reduced level
of EMN in etiolated tissue is not nearly great enough to
account for the very low level of enzyme activity. Ninety-
three xof FMN per gram of dry green leaf tissue is equivalent
to about 1.3 x 10"8 M FMN in the sap from this tissue. In
Table VI, it is shown that over a 10-fold higher concen-
tration of FMN had no effect upon increasing the ig,xit;g
activity of the apoenzyme of glycolic acid oxidase. In
fact, 10-4 mm was necessary for maximum activity in mg
and clearly the leaves ip_ziyg,never contained anywhere
nearly this amount.

Although it has been considered that FMN is the only
prosthetic group for glycolic acid oxidase (66, 67, 69),
other flavins were also tested for possible effects on
enzyme activity. It was found that both FMN and FAD, but
not riboflavin, had an equal effect on the activation of
the enzyme in etiolated cell-free sap from wheat (Figure 2).

Analyses of the FAD sample showed that it was nearly
pure. 0n the other hand, analyses of an aliquot of sap after
incubation with FAD showed that 1 per cent was recovered as
FAD, 90 per cent was present as riboflavin, and 9 per cent
as FMN (Table XI). Therefore, the FAD had been hydrolyzed
by the sap to FMN and riboflavin. Since purified glycolic

41

FIGURE 2
Effect of flavins on enzyme activity

of etiolated wheat sap

zoqi
I6Q— '/////
12d. ' /

~--I—*- FAD or FMN

uptake
(I)
<3
1

cal 02

 

O Riboflavin or none
/ é ,,.-o
<>-*””’

.—~——{%—— 1_,
O’”‘Q j T l I 1

i0 20 30 40 50 80

Time in minutes

V—

Glycolate tollzmoles/flask. Flavins

final concentration I x 10‘4 M.

41

FIGURE 2
Effect of flavins on enzyme activity

of etiolated wheat sap

 

 

 

 

QOqL
16CL— /
0"
12¢.
3‘:
Sam. ---I---- FAD or FMN
D.
:3
(U
<3
,4
3 40.
O Riboflavin or none
g / f
' / /,..o
,,w__43—~ Ace? AC»——"”"
'/;~C*”'§ 1 T 1 1 J
1 2o 30 40 50 60

Time in minutes

Glycolate toIILMOIGB/fIBSk. Flavins

final concentration I x 10'4 M.

TABLE XI

42

Flavins recovered from 1.6 x 10-7 m moles

FAD, after incubation with wheat sap

 

 

 

 

 

Recovery
Flavin
m moles % of total
up 0.02 x 10-7 1
FMN 0.18 x 10‘7 9
Riboflavin 1.80 x 10-7 90
fetal recovered 2.00 x 10'7

 

 

 

Difference between 2.0 x 10’7 and 1.6 x 10‘7 may be

accounted for by the added physiological level of

flavins in the sap or by an error in analysis.

 

43

acid oxidase is not activated by FAD (67), the above effect
from FAD on the enzyme in crude sap could be accounted for
by its hydrolysis to FMN.

Flavins in tissue undergo a dynamic change which
might lead to their synthesis or breakdown. The observation
on the breakdown of FAD has been reported by yeast (101),
anilal tissue (103), and plants (102). The physiological
optimal level of FMN necessary to activate the enzyme in,
zizg cannot be determined from our data. However, the order
> of magnitude of 1 x 10‘8 M FMN in green leaves with an
active glycolic acid oxidase is in striking contrast to a
concentration of 1 x 10‘4 M FMN need ig,1;t;g to activate
the apoenzyme. This comparison suggests that ig,yizg the

formation of the holoenzyme is also an enzymatic process.

Belative Contents of Apoenzzme in Green and Etiolated Tissues

In Table XII are data concerning the relative levels
of apoenzyme in green and etiolated tissues. In order to
obtain this answer, a relatively high substrate concen-

tration and an optimal FMN addition at low concentrations of

the enzyme was used. It was assumed that the surface of the

glycolic enzyme should be saturated with the substrate under
these conditions. The data confirm the results of previous

experiments; that is, a higher percentage increase of 02

uptake in etiolated tissue upon the addition of Optimal FMN

 

TABLE XII

Relative levels of apoenzyme in green

and etiolated tissues

44

 

Q32 (average of 2 experiments)

 

 

Wheat sap Glycolate Glycolate + FMN
Etiolated 21 174
Green 116 370

 

l

 

 

 

Glycolate 50 pcmoles/flask, FMN final concen-

tration 1.0 x 10‘3, 1/5 enzyme concentration

of previous experiments.

45

concentration than in green material, and the total amount

of the apoenzyme, on the basis of Q32, is twofold higher in

green material than in etiolated tissue.

Agtivation of the Enzyme from Ammonium Sulfate Precipitate

When the ammonium sulfate fraction collected between

14 and 22 gme. per 100 ml was dialyzed for six hours, the

enzymatic activities in both etiolated and green tissues
were low (Table XIII). In order to restore the enzymatic
activities in either tissue, an excess of FMN was necessary.
These data show that the loss of physiological FMN during
the dialysis of these fractions from both etiolated and

green tissues resulted in enzymatic inactivity. The green

enzyme preparations as a plant extract before precipitation

and dialysis showed high enzymatic activity.

Activation of the Enzyme in vitro by Incubation with the

figbgtrate
In Figure 3 we have a confirmation of published

data that the enzyme of cell-free homagenate of etiolated

Plants can be activated by incubation in the cold with

glycolate (5). There was no activation of the enzyme when

the plant sap was incubated under similar conditions with

water or with glyoxylic acid or H202 which are products of

glycolate metabolism. H202 showed even an inhibitory effect

on the residual enzyme activity. The activation of the

 

46

TABLE XIII

Ammonium sulfate precipitated enzyme from

green and etiolated wheat sap

 

[-41 02 Uptake/hour

 

 

Kind of tissue Glycolate Glycolate + FMN
Green 40 444
Etiolated 20 96

 

 

 

 

Glycolate 10 ,choles/flask. FMN 1 x 10'4 final

concentration. 02 uptake was calculated from

respiration rate between 5 and 20 minutes.

47

FIGURE 3

Preincubation of etiolated cell-free

wheat sap

Cell-free sap was incubated for 18 hours at
2°C. before Warburg determination in 0.033 M. phosphate
buffer at pH 8.3. Final concentration of glycolate,
glyoxylate or H202 was 0.008 M, when it was used for

incubation. Final concentration of glycolate as a

substrate was 0.024 M.

 

48
FIGURE 3

Preincubation of etiolated

cell free wheat sap

 

1OOI-
80-—
60»—
Glycolate
f}40»~
O.
z I
o“J X/
'4 /
'\20r- // r”* Glyoxylate or buffer
_/ 1 A.
O/o/a/ r...”
10 20 30 40 50 60

Time in minutes

49

enzyme at room temperature is so flow that inactivation can-
cels much of the gain and therefore the phenomenon is best
demonstrated at about 2°C. for the activation and at 30°C.
for'meaaurement of enzyme activity.

The enzyme can also be activated from cell-free sap
when incubated with lactate or ca-hydroxybutyrate which are
natural substrates for the active enzyme (Table XIV). The
effect of the incubation with glycolate is also further
elaborated in this table. The activity of the enzyme in
sap from etiolated leaves which was preincubated with
glycolate for 18 hours at 2°C. increased sevenfold
(Q32, 10 and 70); and in sap from green leaves only 34 per
cent (Q82, 68 and 103). There was no increase in Qge when
FMN was added to the green or etiolated sap which had been
preincubated with glycolate. Incubation of etiolated sap
with lactate or aarhydroxybutyrate increased three to four-
fold enzyme activity (age, 10 and 37 or 35). In fact the
enzyme activity in sap after 18 hours of preincubation with
glycolate, but not with FMN, was equal to that measured

immediately in the sap when 1 x 10"4

M FMN, an excess, was
added. This important point may be interpreted to mean

that all of the apoenzyme had been activated by prolonged
incubation with glycolate alone. Presumably this activation
involved a combination of apoenzyme with the naturally

occurring FMN present in the sap. This natural amount of

TABLE XIV

ng of cell-free etiolated sap after 18 hours

preincubation

5O

 

 

 

 

 

 

Preincubated Warburg
with addition Etiolated Green
Glycolate 1O 68
Buffer alone
Glycolate
+ FMN 53 106
0 70 103
Glycolate Glycolate 58 104
Glycolate
+ FMN 69 106
0 0 3
Lactate Glycolate 37 113
Glycolate
+ FMN 86 146
0 0 3
Ofi-Hydroxy- Glycolate 35 114
butyrate
Glycolate
+ FMN 92 150

 

 

 

 

Incubation conditions and reagent concentrations for

both preincubation and 02 uptake determination were
2M. in Figure 3. FMN final concentration

as specific
was x 10'

 

51

FMN was insufficient to activate the enzyme quickly during
the course of a normal warburg measurement lasting 30 or
60 minutes. Because of these facts this activation is
visualized as a slow enzymatic process requiring glycolate
and capable of proceeding at low FMN concentrations.

The addition of FMN to the sap which had been incu-
bated with lactate or oL-hydroxybutyrate showed toward the
glycolate substrate some further increase of enzyme activity
of about twofold. This enzyme activity was even about 20
per cent higher than that fraction of the same sap pre-
incubated with glycolate. Similar results were obtained
with sap from green material in that preincubation with
lactate or o(-hydroxybutyrate gave more active enzyme
preparations than glycolate incubation when excess FMN was
added to the final assay with glycolate. Without the addi-
tion of FMN, differences among the enzyme activities from
preincubation with glycolate, lactate or a(-hydroxybutyrate
were not appreciable. The somewhat better activation of the
apoenzyme by lactate and oL-hydroxybutyrate than by glyco-
late is hard to eXplain. The rate of the active enzyme
catalyzed oxidation of lactate or ac-hydroxybutyrate is
about one-tenth or less the rate of glycolate oxidation
(5#). Since only one enzyme is supposed to be present for
attack on all three of these oL-hydroxy acid substrates

(6t), these differences in the activation phenomenon should

52

not be caused by activation of other enzymes by lactate and
aL-hydroxybutyrate. Lactate and oc-hydroxybutyrate may serve
to activate the glycolic acid oxidase without appreciable
concurrent oxidation of these substrates. Therefore, these
substrates may remain and be effective longer than glycolate
for the activation. Also rapid oxidation of glycolate by

the activated enzyme may partially inactivate the enzyme,

as for example by the H202 product of the reaction.

The activation of the enzyme in cell-free homogenates
by glycolate cannot be repeated on an ammonium sulfate
precipitated and dialyzed protein fraction which contains
the active enzyme from sap of green leaves (Figure 4, Table
XV). All that is needed for full activity of the ammonium
sulfate precipitate is an excess of FMN which can be added
at the time of preincubation or at the start of manometric
measurement. The Qgé of dialyzed (NH4)2 804 fraction, on
addition of FMN, increased four orimore fold; although this
activity was lower by about one-third than that of the
freshly prepared enzyme from the green tissue, on the basis
of Q32.

Similar, although not identical, results were ob-
tained when the dialyzed ammonium sulfate precipitate from
etiolated tissue was incubated with glycolate (Table XVI).
The loss of enzyme activity on preincubation for 18 hours in

the cold was much higher in etiolated tissue than that in

53

FIGURE #

Preincubation of (NH4)2SO4 precipitate

of green sap

Ammonium sulfate precipitate was prepared as
described in Materials and Methods section. Pre-
incubation conditions were as described in Figure 3.
FMN final concentration was 1 x 10"4 M and that of
glycolate 0.024 M.

For the Warburg assay:

1. Glycolate and 3-glycolate + FMN
after preincubation with glyco-
late.

2. Glycolate and 4-glycolate + FMN

after preincubation with 0.033 M
phosphate buffer.

 

,nal 02 uptake

54
FIGURE 4

Preincubation of (NH4)2804

precipitate of green sap

100
F

 

 

 

 

Time in minutes

 

TABLE XV

qge 0f Preincubated dialyzed ammbnium sulfate

precipitate from green wheat

55

 

 

 

 

 

 

 

 

 

 

 

1 Warburg addition , N

Preincubation Q0
solution Glycolate 2

usmoles FMN
Rene, freshly pre- 24 0 _4 21
pared precipitate 24 1 x 10 143
24 0 8 I:
water 2# 1 x 10‘ 1
24 1 x 10‘4 . 88
O O 20
01 colate 16 0 _ 18
8;.30193 16 1 x 10 4 78
a 0 0 a
1 x 10‘ M FMN 24 ‘ 0
24 1 x 10’4 97
° ° 1 a
1 -4 24 0 0
x 10 M FMN 24 1 x 10.4 '17
glycolate 12 8 13;
o -

1’if?0£ 8M+FMN '5 ‘ 3 1° 4 84

 

burg
Fbr preincubation details see Figure 4. For War
3558! glycolate is listed as/zemoles/flask and FMN

as final concentrations.

 

 

Q52 of preincubated dialyzed ammonium sulfate

TABLE XVI

precipitate from etiolated wheat

56

 

Warbur

ddltlon

 

 

 

 

 

 

 

Preincubation Q32
solution Glycolate FMN
/wmolea/flask Final conc.
one, freshly pre- 24 0 18
ared precipitate 24 1 x 10'4 94
L 24 0 5
ater

24 1 x 10‘4 23

0 O 5

Glycolate 8 ,umoles 15 0 5
16 1 x 10"4 18

O O 5

1 x 10'4 M FMN 24 0 47
24 1 x 10’4 52

Glycolate 8 ,eemoles 0 ° 44
+ 1 x 10'4 M FMN 16 ° 35
15 1 x 10'4 47

 

 

 

 

 

tment see
Fbr details of enzyme preparation and trea ,
Figure 4. warburg experiment is the same as for Table

 

57

green material. When FMN was added to the preincubation
mixture, the loss of enzymatic activity was considerably
reduced and was only about 50 per cent. ~There was no
stimulation of enzymatic activity by glycolate incubation

of etiolated plant sap and again, in order to restore

enzymatic activity, only an excess of added FMN was needed.
The failure to increase the enzyme activity of the
dialyzed (NH4)2SO4 fraction by incubation with glycolate may
be explained by the fact that there was no FMN'in the
dialyzed protein so activation was impossible. Also if the
activation was enzymatically controlled, the protein fraction

may have removed an activating enzyme.

 

SubstratewActivation of the Enzyme in vivo
In Table XVII are data on glycolic acid oxidase

activity in the sap of etiolated wheat seedlings after the
leaves had been sprayed in the dark with glycolate or other
substrates one day before harvest. The an of the enzyme
from plants sprayed with glycolic acid increased fourfold as
compared with plants sprayed with water. These data confirm
a Previous report on the enzyme activation in ziyg_by feed-
ing glycolate (5). When plants were sprayed with lactate,
oL-hydroxybutyrate or glyoxylate it was found that the Q32
0f saps prepared from the sprayed plants also increased
about threefold over plants sprayed with water. Plants

lprayed with acetate did not show appreciable increase

TABLE XVII

Effect of spraying on enzyme activity

of etiolated wheat

 

 

 

 

 

Spraying Qg2
solution ._
Glycolate Glycolate + FMN

Water 10 88
Glycolate 38 104
Lactate 27 104
ofi-Hydroxybutyrate 30 116
Glyoxylate 25 97
Acetate 15 94

 

 

Each flat of plants was sprayed with 10 mmoles of a

compound in 100 ml.

24 hours before harvest.

Plants were sprayed in total darkness

Age of plants at the harvest was

8 days. Control was sprayed with equal volume of water.

For Warburg assay 101/Lmoles of glycolate per flask

were used with 1 x 10" M FMN as specified.

 

59

in enzymatic activity.

FMN was added to the cell-free homogenates from the
sprayed plants in order to measure the total ape-plus holo-
enzyme. There was a many fold increase in total glycolic
acid oxidase activity. However, this activity was nearly
the same regardless of the prior spray treatment. As in
the ig‘zitgg activation by glycolate, the presence of the
enzyme's substrate in yiyg appears to have resulted in the
conversion of all of the apoenzyme to holoenzyme. These
results imply a substrate control of active enzyme activity,

but not over the apoenzyme content.

B. RESPIRATION OF PLANT MITOCHONDRIA

General Experimental Condition Affecting Respiration

The conditions for reproducible maximum rates of
oxidative phosphorylation by pea mitochondria was dependent
upon a number of variables which are discussed in this
section. The system required added ATP, glucose, hexokinase,
and a yeast coenzyme concentrate, but not cytochrome 0.
Optimum substrate and phosphate concentration and duration
of experiments were determined. The order of addition of
substrates and enzymes even influence the reproducibility
of the results.

For active oxidation of citric acid cycle inter-
mediates, ATP, glucose, hexokinase, and yeast coenzyme

concentrate containing DPN, TPN, and CoA must be added to

60

the mitochondria preparation (Figure 5). These requirements
are the same components which are characteristic of

oxidative phosphorylation by animal mitochondria. It has
been reported that P/O ratios, as determined with particles
from tobacco leaves, were not stimulated by added yeast
coenzyme concentrate (80). However, in the absence of the
yeast coenzyme concentrate, pea mitochondria were not capable
of catalyzing the oxidation of the components of the citric
acid cycle. On the other hand, pea mitochondria preparations
contained sufficient cytochrome c that addition of this
component to the assay only slightly increased the rate of
oxidation of citrate or succinate (Table XVIII).

Optimum substrate concentration was determined for
citrate oxidation (Figure 6). A standard amount of mito-
chondria that was obtained from 1.5 g of fresh tissue was
used. The citrate concentration for maximum rate of oxi-
dation was 25 ismoles per 3 ml. of volume in the Warburg
vessel. Although it is impossible to predict from this
finding the optimum concentration of other substrates,
nevertheless, in subsequent studies high substrate con-
centrations were avoided (104) and usually 10 or 20 #- moles
of each substrate per flask were used.

The influence of increasing the phosphate buffer
concentration is shown in Figure 7 for citrate oxidation

and in Figure 8 for malate oxidation. Both oxygen uptake

/:'/. 1 02 uptake

61

FIGURE 5
Effect of ATP-glucose-hexokinase system and
yeast coenzyme concentrate on succinate

oxidation by green pea mitochondria

 

200— ]0
”/f
16dk .' A
,o “- 1
12d»
//
2 ......
8 t" /o
‘1 / 11 /o
/ /’//
// //

1

L«/ 1 ._i_4e~wcr~*T""""Tww' __“J
10 20 3O 40 50 60

Time in minutes
1 - Complete, as described in methods section.

 

2 - ATP-glucose-hexokinase excluded.

3 - Yeast coenzyme concentrate omitted.
All vessels contained 10 LLmOIOB of
succinate. ’

TABLE XVIII

62

Requirements of green pea mitochondria for cytochrome c

and effect of glyoxylate on citrate and succinate oxidation

 

 

 

 

 

 

 

Substrate, ’41 0
1O Asmoles each Cytochrome c Uptak37hr. %
Citrate 0 224 100
Citrate 0.5 mg. 265 118
Citrate + glyoxylate 0 170 76
Citrate + glyoxylate 0.5 mg. 165 62
Succinate O 190 100
Succinate 0.5 ms. 199 105
Succinate + glyoxylate 0 35 18
$Succinate + glyoxylate 0.5 m3. 38 19

 

 

63

Hence» masons: pod canteen no ooaoscix

0.0m -- mmpz: camp mas—é 10%... ..:-mt - -, om

.csmmao noon» no .am m._ Beth
ceasaomu one: means saauoonoouda on» moaosawca
nosemooaoo scene canvases on» confluence madam seem
.no«amuawo easaaao no couuununou ousaamnsm
w Hmblo

[OO—

10m.

1

00w

 

L0mm

anon/bafiidn a0 17?,

64

O O
N c-

O
M

O
43-

Jnoq/peItheise {-Vog.setom1f/

O
in

oopsnsosa nueom eoaoses.

 

 

 

 

 

o.me_ o.eh Oahm m.m.
A A _
to.
m.__u0\m also} +18% ...no\
%, 1,1 1%: ; .iW » 10m
some0111m Jon
. .. -,..;---; w
Joe
esponaeonm
lwom

.easauao no

ceaoacq\om .easpueosm .odpea O\N use eased:
semhxo so soaasapoeosoe openneona ho vacuum

b HmDlo

anon/easidn 30 smoqsvi’

newshound nueom eedoseax

 

 

 

 

 

1: o ..: 11 0 man
1W O—FOoN H O\N mo— ".
m I
0
T.

e

s

m as

J

ft

. LT _

i

u. commune

nu

m. r

.wvo&

o

n

J *

.1ll.nzaonauonm
omw
ow.. .eueasa uo eeaossx ow .opsAaansm
.oaass

0\N use eased: No no coaasaasoosoe easnaeond no vacuum
0 HEDOHE

 

as

 

om

anon/ensidn ao smocsvT/

66

and phosphorylation increased with increasing phosphate
concentration. At higher'phosphate concentration phos-
phorylation increased more than oxygen utilization so that
highest P/O ratios were obtained when an excess of phosphate
was present.

In Table XIX is shown the effect of substrate and
phosphate concentrations and also the length of the experi-
ment on P/O ratios. Highest P/O ratios when malate was
used as a substrate were obtained from short experiment
(30 minutes) with a low substrate concentration (10,znmoles)
and a rather high phosphate buffer concentration.(74-;cmoles).
The P/O in this case was 2.4. However, a P/O ratio of only
1.3 was obtained when the time of the experiment was doubled
and the phosphate concentration halved while retaining the
same substrate concentration.

At the beginning of the measured reaction, one of
the enzymatic systems, mitochondria enzyme or glucose-
hexokinase system, was added from the side arm of the re-
action vessel. Contrary to expectation, this sequence of
additions greatly altered the rate of oxidation. Addition
of the glucose-hexokinase system from the side arm was
greatly superior over incubation with the mitochondria in
the side arm (Table XX). In the former case the mitochondria
was preincubated with a substrate, succinate, for about 45

minutes while the experiment was being set up; 30 minutes at

TABLE XIX

67

Dependency of P/O ratio upon concentrations of

substrate and phosphate and the duration of the experiment

 

 

 

 

 

 

,umoles:
Experiment i P/O
Phosphate Malate
10 1.3
37 20 1.4
40 1.5
60 min. -
10 1.4
74 20 1.8
40 1.7
10 1.9
37 20 1.7
40 1.8
30 min. ‘—
10 2.4
74 20 2.1
40 2.0

 

 

 

 

 

 

TABLE XX

68

Effect of the sequence of addition of reaction

components on oxidation of green pea

mitochondria

 

 

 

 

 

 

 

 

 

 

10‘meoles succin-
ate, 10 /emoles
glyoxylate

 

Reactants in: 02 uptake
* ,al/ hour
‘2‘
Flask Separate side arm
Mitochondria Glucose-hexokinase* 179
10‘;Lmoles succinate
Mitochondria
1O lemoles succinate Glucose-hexokinase 59
+ 10 ;&moles
glyoxylate
Glucose-hexokinase Mitochondria 53
10 femoles succinate
Glucose-hexokinase
10 famoles succinate Mitochondria 54
+ 10 females
glyoxylate
Mitochondria Glucose-hexokinase,
10 pemoles succin- 87
ate
Mitochondria Glucose-hexokinase, 62

 

a In one solution.

** Average of two experiments.

 

69

0-2° during the flask preparation and then 15 minutes at a
temperature up to 30°C. during equilibration and combining
of the reactants. Perhaps during this period the substrate
stabilized the mitochondrial preparation.

Thus, the enzymatic activity of mitochrondria de-
pends upon many factors and a further important variation
was the procedure for mitochondria preparation. Thus, exact
duplication of results was difficult. Still another reason
for reporting P/O ratios over a range of values for the same
substrate was caused by the accuracy of the analytical
methods for 02 uptake by Warburg's method and for esterified
phosphate by Fiske-Subbarow colorimetric procedure. These
methods are unreliable for low values of 02 uptake and
phosphate esterified. Unfortunately,'there are numerous
reports in the literature for P/O ratios based on values of
one or less inmoles of oxygen or phosphorus (80).

Isolated mitochondria from both green and etiolated
Pea leaves catalyzed rapid 02 uptake and orthophosphate
esterification during the oxidation of the acids of the
citric acid cycle (Table XXI). The P/O ratios obtained con-
firm similar data now in the literature. Oxaloacetate was
the only acid for the citric acid cycle which was not

Oxidized by my mitochondrial preparations.

TABLE XXI

Oxidative phosphorylation of pea mitochondria

70

 

 

 

 

xiatoms Oe/hour* P/O
Substrate,
20 females

Green Etiolated Green Etiolated
Citrate 12.6 10.0 2.7 3.2
Cis-aconitate 4.0 -- 1.8 --
Isocitrate 11.9 I -- 1.4 --
Oxalosuccinate 5.1 -- 1.7 --
oL-Ketoglutarate 31. 9 -- 2. 0 ~-
Succinate 15.4 7.8 1.7 , 2.0
Funerate 5.9 -- 2.1 --
Malate 7.2 -- 3.1
Oxaloacetate 0.0 -- 0.0 --
Glycolate 8.0 0.0 0.3 0.0
Glyoxylate 0.0 0.0 0.0 0.0
Glyoxylate 2.1** I -- 1.3 --

 

 

 

 

 

* Mitochondria from 0.66 - 1 gm. tissue.

** Mitochondria from 2 - 3 gm. tissue, average from 6

experiments.

 

71

Oxidation of Glycolate and Glyoxylate by Mitochondyia
Glycolic acid was also rapidly oxidized by mito-

chondrial particulates. Glycolic acid oxidase is considered
to be a soluble enzyme of the cytoplasm. Glycolate oxidation
could have been catalyzed by this enzyme located in the
mitochondria themselves or by the absorption on the mito-
chondrial surfaces of the cytoplasmic glycolic acid oxidase
during their isolation. It is well known that mitochondria
possess such absorptive abilitives and even washing of
particulates several times does not remove absorbed enzyme
(80, 81).

Although glycolic acid was rapidly oxidized, there
were only traces of orthophosphate esterified and the P/O
ratio was practically zero as Zelitch has also shown (80).
It is concluded that the oxidation of glycolic acid by
mitochondria is different in character from that involved
in the oxidation of the constituents of the citric acid
cycle acids. These experiments had been initiated to deter-
mine whether the FMN dependent glycolic acid oxidase system
could be coupled to the cytochrome electron carriers as is
the FAD succinic acid dehydrOgenase system. The low P/O
ratio of 0.3 indicated that this coupling of glycolate
oxidation to oxidative phosphorylation was not feasible.

The oxidation of glyoxylate was extremely slow, and
in fact, the oxidation of glyoxylate could only be

72

demonstrated by increasing the amount of mitochondria three
to fivefold. Hewever, there was a reproducible and sub-*
stantial P/O ratio of greater than one accompanying this
oxidation after correction for endogenous effects. The
explanation for this phosphorylation has not been elucidated.
The most likely explanation is that some malate synthetase
was present in the mitochondrial preparations. Malate
synthetase has been shown to be very active in mitochondria
preparations of five-day old pea cotyledons (58), and it is
also present in much lower but measurable amounts in mature
leaves of other plants. In our case the mitochondria were
prepared from eight to ten-day old pea leaves and, therefore,
malate synthetase was very likely still active in such
preparations. This enzyme would have condensed glyoxylate
with endogenous acetate to produce malate whose subsequent

oxidation would account for the P/O values.

Inhibition of Oxidation by Glycolate or Glyoxylate
Both glycolate and glyoxylate inhibited the oxi-

dation of the acids of the citric acid cycle by mitochondrial
preparations. .The inhibition consisted of a reduction in
the rate of oxygen uptake and an uncoupling of oxidative
phosphorylation for that part of the oxygen uptake which was
not inhibited. Data from the use of glycolate as the in-
hibitor was difficult to interpret due to the oxidation of

73

the glycolate itself as discussed in the previous section.
This difficulty was surmounted by using glyoxylate which was
not oxidized at an appreciable rate or by using mitochondria
which had been prepared from etiolated plants since they
contained little glycolic acid oxidase.(34).

The effect of glycolate and glyoxylate on the in-
hibition of mitochondria respiration by each substrate of
citric acid cycle has been studied in detail. The effect
upon citrate and succinate oxidation by mitochondria from
green peas are shown in Tables XVIII, XX, and XXII and
Figure 9, the effect upon the oxidation of malate is shown
in Figure 10, and for the effect on oxalosuccinate and

oc-ketoglutarate oxidation see Figure 11. The total oxygen
uptake in the presence of glycolate was greater than that
for citric acid component alone, but much less than the sum
of the oxygen uptake for the citric acid component and
glycolate separately. These results were not caused by an
insufficiency of oxygen to permit both systems to function
at maximum rates. Dilution of the mitochondrial prepa-
rations did not alter the ratio of the results. Further
glyoxylate also inhibited the oxidation although there was
no significant oxygen uptake from the glyoxylate alone.
Data on the P/O ratio from glycolate plus a citric acid
cycle compound is difficult to interpret because it consisted

of two components which could not be differentiated. One

TABLE XXII

74

Effect of glycolate and glyoxylate on citrate and succinate

oxidation by green pea mitochondria

 

 

 

 

 

 

 

 

 

Oxygen uptake Phosphorylation
Substrates,
10 ,amoles ’
each xaatoms
02/hour % E/O %
Eeyigent A
Citrate 7.0 100 2.2 100
Glycolate 8.0 114 0.3 14
Glyoxylate 2.1 30 1.3 59
Citrate + ‘
glycolate 8.9 127 0.7 32
Citrate +
glyoxylate 5.4 77 1.8 82
Experiment B
Succinate 6.7 100 2.7 100
Glycolate 8.7 130 0.1 4
Glyoxylate 0.5 9 0.0 0
Succinate +
glycolate 11.0 164 0.8 30
Succinate + #1
glyoxylate 4.0 60 1.1

 

 

75

FIGURE 9

Effect of glyoxylate on citrate and succinate
oxidation by green pea mitochondria.

1 - Citrate.

2 - Citrate + glyoxylate.

3 - Succinate.

4 - Succinate + glyoxylate
Substrates, 20 ;Amoles each. The results with
citrate and succinate substrates were determined

in two separate experiments.

[rel 02 uptake

76

FIGURE 9
Effect of glyoxylate on citrate and
succinate oxidation by green pea

mitochondria

3001—

250*—

200-

150 —

100-

 

 

 

Time in minutes

/zal 02 uptake

77

FIGURE 10
Effect of glycolate and glyoxylate on
malate oxidation by green pea

mitochondria.

125r

 

 

 

9
//
/,
/
100— /
o L 2
o
75*-
L .
SO*'
25—
o
l i l L l
10 20 30 4O 50 80

Time in minutes
1 "' Malate, P/O = 3e'e
2 - Malate + glycolate, P/O = 0.4.

3 - Malate e glyoxylate, P/O = 1.5.
Substrates, 20/zsmoles each/flask.

’fibl 02 uptake

78

FIGURE 11
Effect of glyoxylate on oxalosuccinate

and oo-ketOglutarate oxidation by green

pea mitochondria.

160

120

~-
~

 

 

3O 4O 50 60
Time in minutes

1 - Oxalosuccinate, 2-oxalosuccinate + glyoxylate.
3 - at -Ketog1utarate, .4 oL-ketoglutarate e glyoxy- -
late. Substrates, lo/asmoles each. Two separate
experiments were run for each substrate, oxalo-

succinate and oc-ketoglutarate.

79

was the oxidation of glycolate itself with a low P/O ratio
and the other was the inhibited oxidation of the citric
acid substrate. Consequently, very low P/O ratios were ob-
tained from such a system because that portion of the
oxidation attributed to glycolate contributed no energy for
the phosphorylation.

One way the complexities of the above results are
simplified was to use mitochondria that had been isolated
from etiolated peas. The activity of glycolic acid oxidase
is in the order of tenfold less in etiolated tissue (5, 34),
and thus the contribution of glycolate oxidation in the com-
bined system was much less. Glycolate inhibited 50 per cent
the rate of oxygen uptake for citrate and succinate oxi-
dation by mitochondria from etiolated peas (Table XXIII).
However, for the remaining oxidation, the P/O ratio was not
reduced significantly. These results suggested that the
effect of glycolate was primarily in inhibition of the
initial part of citrate oxidation rather than any effect
upon the cytochrome electron transport process.

A second way to simplify the studies of the in-
hibitory effects of glycolate and glyoxylate was to utilize
only glyoxylate with sufficient mitochondria to obtain
measurable oxygen uptake with the citric acid cycle com-
Ponents but not with glyoxylate. Then the correction for

respiration with glyoxylate alone, which was always used for

 

TABLE XXIII

80

Effect of glycolate and glyoxylate on citrate and succinate

oxidation by etiolated pea mitochondria

 

‘

 

 

 

 

 

 

Oxygen uptake Phosphorylation
Substrates,
20 ,4. moles
each atoms
2/hour % P/O % ‘
Experiment A
Citrate 12.0 100 3.0 100
Glycolate 0.0 0 -- --
Glyoxylate 0.6 5 -- --
Citrate +
glycolate 5.9 49 2.9 97
Citrate +
glyoxylate 6.2 52 2.5 83
Experiment B
Succinate 22.6 100 1.6 100
Glycolate 0.2 1 -- --
Glyoxylate 0.8 4 -- --
Succinate +
glycolate 12.3 54 1.4 88
Succinate +
glyoxylate 4.0 19 1.3 81

 

 

 

81

calculating the P/O ratio, was not large and could not intro-
duce great uncertainties. Glyoxylate was_at least as potent
an inhibitor as glycolate or better. With mitochondria from
green peas the glycolate was in fact oxidized to glyoxylate
and so it was not possible to know which acid was the true
inhibitory, but glyoxylate was a better inhibitor (Table
XXIII and Figure 12). Glyoxylate (0.007 M final concen-
tration) inhibited succinate oxidation 80 per cent. With
mitochondria from green peas very substantial inhibition was
also obtained with glyoxylate (Tables XXII, XXIII, XXIV,
Figures 9, 10, 11). In general, the glyoxylate inhibition
did not reduce the P/O ratio of the remaining respiration
as much as glycolate because the glyoxylate itself was not
oxidized. Nevertheless, there was always a very measurable
reduction of the P/O ratio in the presence of glycolate.
Zelitch and Barber (80) reported no lowering of the P/O
ratio from glyoxylate inhibition of succinate and citrate
oxidation by mitochondria from spinach. We are not certain
about the importance of this difference between plant
tissue, because the magnitude of the lowering of P/O ratio
in our system with glyoxylate was not always large nor

consistent.

Consideration of "Oxalomalate" as Inhibitor
As discussed in the literature review section,

Ruffo's group, using liver homogenates, reported on glyoxylate

nt‘1 02 uptake

82

FIGURE 12
Effect of glyoxylate on citrate and
succinate oxidation by etiolated

pea mitochondria.

12G5

80*

 

 

 

! 1 1 l_J 1 _TJ
10 2O 3O 4O 50 60

Time in minutes
1 citrate, 2 citrate e glyoxylate,
3 succinate, 4 succinate + glyoxylate.
Substrates, 20/LLmoles each. Two separate

experiments were run for each substrate.

 

TABLE XXIV

83

Oxidation of oxaloacetate by green pea mitochondria

 

 

 

 

 

/u.1 02 uptake

Substrate, 10 __ __
;4moles each I I

10 min. 25 min. 45 min. 60 min.
Oxaloacetate 0 O O 0
Glyoxylate 3 11 13 18
Succinate 27 68 136 188
Glycoxylate +
succinate 15 33 43 52
Glyoxylate +
oxaloacetate 1 { 1 9 . 13 )

 

 

 

 

 

After respirometric measurements, it was found that 73% of

oxaloacetate unreacted when it was incubated alone, and

53% of it remained when it was incubated together with

glyoxylate.

84

inhibition of oxidation of the citric acid cycle components.
This inhibition was particularly severe when both glyoxy-
late and oxaloacetate were added to the incubation mixture
and simultaneous accumulation of citrate had been observed.
That group postulated that the inhibition was caused by the

hypothetical compound "oxalomalic acid"

which could have been formed by a condensation of glyoxylic
acid and oxaloacetic acid. Since oxalomalate has a con-
figuration similar to citrate, it could have inhibited
citrate oxidation as a competitive inhibitor for aconitase.
Our results differ from the observations from
Ruffo's group and consequently we do not find their ex-
planation of glyoxylate inhibition satisfactory for plant
mitochondria. For mitochondria from peas glyoxylate in-
hibition was not limited to citrate oxidation, but it was
also severely effective on the other substrates of the
citric acid cycle. Fer sL-ketoglutaric acid oxidation, for
instance, there was no lag in the rate of this inhibition at
the beginning of the experiment (Figure 11). It is unlikely
‘that a specific inhibitor of aconitase could instantaneously

affect, at a constant rate, the oxidation of a large excess

85

of oc-ketoglutaric acid, succinate, or malate.

Oxaloacetic Acid and oz-Ketoglugaric Acid Oxidation

Added oxaloacetate was not oxidized by our pea
mitochondria even in the presence of acetyl phosphate, co-
enzyme A and transacetylase (Table XXIV). Analysis for
oxaloacetate from incubation for an hour with the mito-
chondria, revealed that, despite this compound’s known
instability, 73 per cent of it was still present in the
system.

When malate-C14 was oxidized by pea mitochondria
only traces of C14 appeared in citrate during the periods
of time normally used to determine P/O ratios. Apparently
the Observable oxidation was mainly accounted for by steps
before citrate formation.

These data in oxaloacetate oxidation need further
investigation. Apparently the condensing enzyme system was
severely restricted in the mitochondria as isolated. The
data supports the contention that the glyoxylate inhibition
of malic and succinate oxidation cannot be explained on the
basis of an effect only on aconitase by "oxalomalate."

The inhibition by glyoxylate of’aL-ketoglutarate
oxidation was not as severe as the inhibition by malonate
(Figure 13). However, the inhibitory effect from both

glyoxylate and malonate was nearly additive. Since malonate

86

FIGURE 13

Single step oxidation of az-ketoglutarate

120

/u’1 02 uptake

 

 

J J _,
40 50 60

Time in minutes

 

1 oL-ketoglutarate, 2 cL-ketoglutarate 4-
glyoxylate, 3 aL-ketoglutarate e malonate,
4 ot-ketoglutarat'e a malonate + glyoxylate.

Malonate alone was not oxidized. Substrates,

10 [smoles each/flask.

87

is known to block succinate oxidation, the inhibition by
glyoxylate would have to be accounted for by an inhibition
of the steps before malate formation. This argument would
support our contention that glyoxylate inhibition is at
least not all accountable for by an effect on aconitase.
Oxaloacetate was an inhibitor of mitochondrial
oxidation of other components of the citric acid cycle
(Table XXV). Oxaloacetate inhibited citrate oxidation by
55 per cent and oL-ketoglutarate oxidation by 25 per cent.
The inhibition from oxaloacetate and glyoxylate combined

was additive and consequently very severe.

§§imulation of Succinate Oxidation by Glyoxylate and
and Inhibitign by Thioglycolate

Whereas, glyoxylate in equimolar concentrations with

succinate inhibited succinate oxidations low concentrations
Of glyoxylate produced as sustained stimulation of succinate
oxidation (Figure 14). Careful measurements of oxygen up-
take when 10 pmoles of succinate and 10 ,u-moles of glyoxy-

late were present showed an initial stimulation of

resPiration followed by severe inhibition. After one hour

succinate oxidation was inhibited by 48 per cent. When the

glyoxylate concentration was only one‘ptmole, however, the

initial stimulation of succinate oxidation was maintained for

at least an hour during which time there was recorded a

TABLE XXV

Effect of glyoxylate and oxaloacetate on citrate and

ot-ketoglutarate oxidation by green mitochondria

88

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Oxygen uptake
Substrate, 10 min. 25 min. 45 min.’60 min.
Exp. 20 “moles _
each ,
Citrate 25 100 72 100 145 100 184 100
Oxaloacetate (0AA) 2 6 5 ‘ 3
Glyoxylate 16 18 19
1 0AA + glyoxylate 4 8 12 10
' Citrate + glyoxylate 23 92 65 90 123 85 148 80
Citrate + 0AA o 0 14 19 501 34 65 35
Citrate + glyoxylate
... GM 3 12 11 15 21 14 21 11
at-Ketoglutarate 56 1100 1511100 277 100 352 100
0AA 0 0 O 0
Glyoxylate 5 2 15 15
2 0AA + glyoxylate O 0 4 6
at-Ketoglutarate + OAAj 30 54 85 56 195 70 265 75
I
ac-Ketoglutarate +
glyoxylate 3O 54 58 45 141 51 177 50
ol-Ketoglutarate + 1
glyoxylate + 0AA 8 14 181 12 65 23 86 24

 

 

 

FIGURE 14
Effect of various glyoxylate concen-
trations on oxidation of succinate by

green pea mitochondria.
2007—

. 4C1
160— {x/

 

 

 

 

120— ',°
1 z//
3 /
3 / ’ 1, ./ 3
9 ,, J 1— ,0
Am 80" /////Q ,/‘ rr///////
_ ‘ --2
93.: / ' F ,3 .Q
,0 -~/""/
/ / /F/ -
40 e / o
O /
.2 8
[/1 , / v
,%///_AJ‘M,.MJ 1 J 1 _ _ 1
1O 20 3O ‘40 50 50

Time in minutes

0
d
I

Succinate 10_;Lm.

2 - Succinate Io/zzm e glyoxylate Io/zcm.
3 - Succinate 10’;Lm e glyoxylate Slle.
4 - Succinate 10 men a glyoxylate 1 /Lm.

 

90

total of 39 per cent stimulation of respiration by glyoxy-
late. Thioglycolate has often been used to protect enzymatic
systems because of its -SH component, but-its use with the

plant mitochondria was most inhibitory (Table XXVI).

Stimulation of Glyoxylate and Succinate Oxidation by FMN
Addition of FMN to green pea mitochondria stimulated

 

several fold either glyoxylate or succinate oxidation (Table
XXVII). The reason for either of these stimulations is not
clear. Glyoxylate reduction by glyoxylic acid reductase
would require DPNH (75) and FMN could only indirectly affect
the system by stimulating the reoxidation of glycolate by
glycolic acid oxidase.

Glyoxylate utilization by isocitrase should not be
stimulated by FMN. Glyoxylate oxidation to oxalate by
glycolic acid oxidase (73) would be stimulated by added FMN.
1 x 10"2 molar FMN stimulated succinate oxidation 100 per
cent. Although this is a high concentration it is not
indicative of the concentration inside the mitochondria.
Succinic dehydrogenase from yeast bacteria and animals have
FAD as a prosthetic group. The nature of flavin in succinic
dehydrogenase from plant tissue has not been established.
Though data suggests the possibility that succinic dehydro-
genase might be an FMN linked system in plants, other ex-

Planations for the data are possible.

91

TABLE XXVI

Effect of thioglycolate on succinate oxidation

by green pea mitochondria

 

 

 

 

 

 

02 uptake
Substrate ,ul/hr. 7!

1O ,umoles succinate 76 100
10 [z moles glyoxylate 18
50 females thioglycolate 30
50 [emoles thioglycolate + 10 fimoles

glyoxylate 23
10 #moles succinate + 10 ”moles '

glyoxylate 54 71
10 #moles succinate + 50 pmoles

thioglycolate 36 47
1O ,umoles succinate + 10 #4110168

glyoxylate + 50 pmoles thio-

glycolate ' 26 34

 

 

92

TABLE XXVII

Effect of glyoxylate and FMN on succinate

oxidation by green pea mitochondria

 

 

 

 

 

FMN ;L1 02
substrate, 10/smoles each Final Conc. Uptake/hr.
0 4
1 x 10"2 28
Glyoxylate 1 X 10"3 32
1 1 x 10‘4 29
O t 79
1 x 10'"2 160
Succinate 1 x 10'3 132
1 x 10" 93
O 46
1 x 10"2 73
Succinate + glyoxylate 1 x 10"3 66
1 x 10'4 41

 

 

 

 

There was no oxygen uptake with FMN alone.

93

Interppactions Betwgen Succinate, Glyoxylate and Glutamate

For considerations about any regulatory control of
citric acid cycle oxidations by glyoxylate, the effect of
associated reactions of glyoxylate which control the avail-
ability of glyoxylate would be pertinent. One such set of
reactions of glyoxylate is its conversion to glycine in the
presence of the glyoxylate-glutamic acid transaminase system.
In our present investigations glutamate was oxidized by the
pea mitochondria, presumably via.ct-ketog1utarate (Table
XXVIII). The oxidation of succinate and glutamate was
additive as expected and glyoxylate inhibited succinate
oxidation as previously observed. Glyoxylate stimulated
glutamate oxidation probably because it accelerated its
conversion to oL-ketoglutaric acid by the transaminase re-
action. Addition of glutamate to the glyoxylate inhibited
succinate system stimulated the respiration fourfold. Though
an exact interpretation of so complex a system as the latter
is not possible, the results indicate that the expected
interactions among the substrates were occurring. Addition
of glutamic acid resulted in stimulated respiration due to
formation of oc-ketoglutarate and probably to removal of the

inhibitory glyoxylate.

Effect of Glyoyylate and Cysteine on Mitochondria; Respiration

One way in which glyoxylate might inhibit the

TABLE XXVIII

Effect of glyoxylate and glutamate

on succinate oxidation

94

 

/¢l.02 uptake

 

 

 

 

‘

 

 

 

 

Substrate, _r
10/zmoles each min. 10 min. 25 min. 45 min.l60 min.
Succinate 25 44 92 1 15 194
Glutamate 9 14 32 58 75
Glyoxylate 5 6 8 15 14
1Succinate + gluta-
mate 1 32 54 120 210 255
Succinate + glyoxy1 .

. late 14 21 36 52 57
Glutamate + glyoxy~

1 late 19 38 86 151 190
Succinate + gluta-

‘mate + glyoxylate 25 47 105 181 231

 

 

.1.

1.?

win..-

 

95

mitochondrial oxidation of the acids of the citric acid
cycle was through a reaction of its addehyde group with an
éSH site which was necessary for enzymatic activity. We
might, therefore, expect -SH compounds to protect against the
inhibitory action of glyoxylate. Such was not the case with
thioglycolate (see previous section) (Table XXVI). However,
a fivefold excess of cysteine did prevent glyoxylate in-
hibition of succinate oxidation (Table XXIX), but not of
malate oxidation. Cysteine by itself, like thioglycolate,
was inhibitory to mitochondrial oxidation of succinate. The
failure of cysteine to prevent glyoxylate inhibition of
malate oxidation excludes the possibility of a cysteine and
glyoxylate interaction and in this way the removal of

glyoxylate.

EffectqofwTyposinase apgffiscorbic Acid Oxidase

Since glycolate and glyoxylate are the substrate and
product of a terminal oxidase, it was conceivable that other
terminal oxidase systems might inhibit mitochondrial
respiration. Tyrosinase was absent in the pea mitochondrial
Preparations (Table XXX) and ascorbic acid oxidase activity
was low even in comparison to the activity of the glycolic
acid oxidase. Substantial amounts of all three of these
oxidases were present in the cytoplasm. The oxidation of

succinate in the presence of either tyrosine and DOPA or

TABLE XXIX

96

Reversal of glyoxylate inhibition of succinate oxidation

by cysteine

 

 

 

 

 

 

02 uptake
Substrate )sl/hr. %

1O ztmoles succinate 139 100
10 [smoles glyoxylate 14
5O ;smoles cysteine 2O
50 zcmoles cysteine + 10 famoles

glyoxylate 7
1O pcmoles succinate + 10 femoles

glyoxylate €54 39
I10 fi-moles succinate + 50 ”moles

cysteine 92 66
1O femoles succinate + 10 /¢moles

glyoxylate, + 50 [emoles cysteine 134 96

 

 

TABLE XXX

97

Effect of glyoxylate, tyrosine, 3,4-di-hydroxyphenylalanine

(DOPA), and ascorbic acid on oxidation of succinate

 

_

 

 

 

 

 

 

 

Substrate, ;al 02 uptake
1 10 memoles 4—
each
10 min. 25 min. 45 min. 60 min.
eriment_1
Glyoxylate 2 6 11 11
Tyrosine 0 O 2 O
1 DOPA 0 0 5 5
Succinate 26 68 130 166 1
Succinate +
glyoxylate 23 46 7O 84
1 Succinate +
1 tyrosine 31 73 121 150
Succinate +
DOPA 30 70 110 135
Agaperiment 2
Glyoxylate 0 3 11 13
Ascorbate 12 17 34 41
Succinate 37 74 136 174
Succinate +
glyoxylate 31 48 76 88
Succinate +
ascorbate 75 134 176

 

 

 

98

ascorbate was not substantially suppressed. These results
indicate that the effect of the glycolate—glyoxylate terminal
oxidase system on inhibiting mitochondrial respiration was

different from the other terminal oxidases.

In Vivo Inhibition of Respiration by Glyoyylate
If glyoxylate were inhibiting mitochondrial

respiration, then feeding plant tissue glyoxylate should
result in an inhibiting of respiration pp,yiyp (Table XXXI).
Normally the amount of glyoxylate present in leaves is
extremely small. Though much carbon from the photosynthetic
carbon cycle moves through a pathway going from glycolate to
glyoxylate, to glycine, to serine, to glyceric and then to
sugars, the pool size of the glyoxylate is so extremely small
that seldom is any glyoxylate-C14 detected on chromatograms.
The significant inhibition of respiration pp,yiyp by
glyoxylate suggests that if it were to accumulate in leaves
it would be inhibitory.

Nature of the Glyogylate Inhibition
The Lineweaver-Burk plot for the effect of glyoxylate

inhibition of succinate oxidation by green pea mitochondria
indicates that the glyoxylate inhibition was noncompetitive
(Figure 15). The fact that glyoxylate inhibited the
oxidation of all the components of the citric acid cycle is
an additional reason for‘believing that this glyoxylate effect

was not caused by substrate similarity with the other acids.

99

TABLE XXXI

Effect of glyoxylate on citrate and

succinate oxidation 1 vivo

 

 

 

 

 

 

 

 

02 uptake

substrate, 20 famoles each gal/hr. %

O 156 100
Glyoxylate 146 94
Citrate 137 88
Succinate 131 84
Citrate + glyoxylate 95 61
Succinate + glyoxylate 122 78

 

0.5 g. of 1 cm. long segments of the uppermost part of
10-day old green pea seedlings were placed in each Warburg
vessel containing 37 zemoles of phosphate buffer at pH

7.3,total volume of reactants was 3 ml.

100

FIGURE 15

Inhibition of various levels of succinate oxidation by

glyoxylate

Enzyme source was prepared from green pea mito-
chondria as described in the Materials and Methods
Section. Standard manometric assay was used. Three
levels of succinate, 10, 20, and 30 zcmoles were used
as a substrate and 20 ,amoles per flask of glyoxylate
as an inhibitor. S represents molarity of succinate

used and v /catoms of 02 taken up per hour.

101

 

 

 

00¢
_

oasHmKOhHw a

P
—
COM OON OO- - 111%. 11111111111111.1110”. .1..\.1v..1 11111
41 d — 1111111111111 1 \\.\
cessaoosm.1 1\\\\\\\\\1\1\\\\\\\1 \\\
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me

102

Rgverspl p: the Glyoxylate Inhibition by DPN
In.the study of a citric acid cycle intermediate

oxidation by mitochondria preparations, the first product
formed is usually also further metabolized and the results
‘recorded as 02 uptake are not necessarily due entirely to the
oxidation of the added substrate. In order to eliminate this
complexity pure malic dehydrogenase was used and the inhibitory
effect of glyoxylate on malate oxidation was studied.

In Figure 16 are data representing the effect of
glyoxylate on the malic dehydrogenase system. The results
were recorded as the optical density increase at 340 mzL in
a Beckman DU spectrophotometer due to a reduction of the added
DPN. Optimum final concentration of DPN was 0.003 M and
0.001~M.DPN was also nearly an optimum concentration for 25
units of the enzyme. When.0.05 M final concentration of
Glyoxylate was added to the cuvette containing.0.003 M DPN,
there occurred a 39 per cent inhibition of malate oxidation
and in the presence of 0.001 M DPN this inhibition was 56
per cent. Upon the addition of DPN in excess (0.02 M), no
inhibition of malate oxidation by glyoxylate was obtained.

Similar effects upon the inhibition of lactic acid
dehydrogenase by glyoxylate were obtained and again excess
of DPN prevented the inhibition (Figure 17). Because the
equilibrium constant is very unfavorable for the reaction

lactate-————4>jpyruvate (109), a high concentration of

103

FIGURE 16

Glyoxylate inhibition of malic dehydrogenase

1 - DPN, 0.001 M

2 - DPN, 0.003 M

3 - DPN, 0.001 M + glyoxylate, 0.05 M

4 - DPN, 0.003 M + glyoxylate, 0.05 M

5 - DPN, 0.02 M + glyoxylate, 0.05 M
All cuvettes contained: glycine, 0.1 M; malate,
0.01 M; and malic dehydrogenase, 25 units. Final

pH was 10.5. Reagents are listed as final con-

centrations.

Optical density

0.150-

_.__Lillnw _. -i.1.l-_ ..
4 8 12 16

104

FIGURE 16

   

l...__._._

Time in minutes

Optical density

0.200

0.150

0.100

105

FIGURE 17
Glyoxylate inhibition of lactic acid

dehydrogenase.

 

I
..—
1

T

 

 

 

[/g .1- ..1..--- “-21-“--l_i_l1 _ . 1
6 8 10

2 4
Time in minutes

1 - 0.003 M DPN (optimal).
2 - 0.003 M DPN + 0.15 M glyoxylate.
3 - 0.05 M DPN e 0.15 M glyoxylate.

All curettes contained: Lactic dehydro-
genase, glycine 0.1 M, and lactate 0.1 M.
Final pH was 9.9. All reagents are
listed as final concentrations.

"7‘". T“ 251.6“.‘1‘2‘5‘ 1
's

Wasn't? .. .1” t“:

106

substrate had to be used. A linear reaction with time was
obtained for only four minutes, probably because of an un-
favorable equilibrium and product inhibition (104).

To explain the above results we have considered the
fact that both glyoxylate and DPN are reported to be able to
form complexes with other compounds. Glyoxylate condenses,
for example, with tetrahydrofolate (105) and DPN with
glycolaldehyde (107) or dihydroxyacetone (106). All of
these condensations are non-enzymatic. In an attempt to re-
veal a condensation product of glyoxylate-DPN a different
absorption spectrum from either oxidized or reduced DPN was
sought by means of a Beckman DK-2 recorder. No change in
the DPN spectrum in the region of 340 m;c was observed.
Measurement at lower wave lengths was obscured by the dis-
covery of a condensation of glyoxylate and the glycine
buffer at pH 10.5 (Figure 18). The presence of such con-
densation products were confirmed chromatographically

The explanation for glyoxylate inhibition of DPN

 

linked dehydrogenases then becomes a question whether
glyoxylate or a glyoxylate-glycine complex causes inhibition
of substrate oxidation perhaps by removing the DPN from the
enzymatic reaction. In order to answer this hypothesis
other buffers than glycine were used. When carbonate-bi-

carbonate buffer was used, the inhibition of malate

% Absorbancy

FIGURE 18
Non-enzymatic condensation of glyoxylate
and glycine, at pH 10.5, as reproduced
from Beckman DK-2 chart.

 

 

 

107

 

 

 

'00 1‘ 1 x
1
1 \y
\\
805
' 1
60— 1
. 1 \
1 1 \
40% \. \\
I -—->-1\\«<—~2 \\ +— 3
1
1
20%-
1i \ \
1 \\ \\\\ \.
i \ \_:\$\\\\\k \\\\\-
A - _ ___. Ji 7 L \if“ ‘r 1
220 240 260 280 300

Wavelength, 101»

320

1 - Glyoxylate, 0.01 M, in water or 0.1 M carbonate-

bicarbonate buffer.
2 - Glycine, Oe‘ Me

3 - Glyoxylate, 0.01 M e glycine, 0.1 M, combined
together for 5 minutes.

All reagents are listed as final concentrations.

Non-enzymatic reaction of 0.1 M glycine with 0.1 M

FIGURE 19

glyoxylate at room temperature and pH 10.5.

1.2.3
4.5.6

 

----m—- Phenol
Spots when glycine was chromatographed.

‘Glycine + glyoxylate reacted for 15 minutes.
Methyl red dye.

Methyl orange dye.

No spots detected with glyoxylate alone.

108

Butanol-propionic acid ——s~——~

O

109

oxidation by glyoxylate and the reversibility of this in-
hibition by excess of added DPN was essentially the same as
when glycine had been used as the buffer (Figure 16). Hew-
ever, no change in the absorption spectrum for glyoxylate was
found when the reaction was run in water or in carbonate-
bicarbonate buffer (Figure 18). These results show that the
condensation product of glycine and glyoxylate was not
responsible for the inhibition of the enzymatic reaction.
The ultraviolet absorption spectra of a solution of
DPN or DPN 4 glyoxylate were essentially similar. Conden-
sation of glyoxylate might have occurred with DPNH at the
C4 position of the nicotinamide ring without shifting the
double bonds within the ring so that the final spectrum
would be similar to that of reduced DPN. The following

reactions would occur according to this hypothesis:

 

 

F coon '1
NH H ..c!
H H l 2 -1\1H2
l C: O _ C :0
1 4’ 00011 ___> 1 1
1‘1 .0110 N
11
R Glyoxylic
DPNH Acid " 4 J

110

p 0 ._ 1p 0 1
ll Al
H—(C\N—H ._ / \ N
é=o '1... e.
.__4>. -——-€>
| l 4 H20 ' |
N N
1. 1!.
b A L. -4

 

 

 

 

As a result glyoxylate would inhibit DPN catalyzed reactions
either by removal of the DPNH or by formation of an inhi-
bitory complex of DPNH.

This hypothesisxhas been supported by chromato-
8raphic assays of DPNH and glyoxylate mixtures and detection
of the DPN component by 36604A° ultraviolet light. Ascend-
ing single dimensional chromatography was used with a sol-

vent of the following composition: 600 grams of ammomium

111

sulfate were dissolved in one liter of 0.1 M sodium phosphate
buffer, pH 6.8, and 20 m1 of n-propanol were added. DPN and
glyoxylate chromatographed as the DPN control which was a
dark blue absorbing spot at Rf of 0.31. Apparently DPN and
glyoxylate did not react. DPNH chromatographed with an R1.
of 0.12 and showed a light blue fluorescent color. With
DPNH plus glyoxylate a new spot appeared at the Rf of 0.19
which was also light blue fluorescent in color. Apparently
DPNH and glyoxylate had formed non-enzymatically a complex
with the same ultraviolet fluorescent characteristics as
DPNH. That it was not DPNH but a new compound was known from
its chromatographic properties. It is proposed that this
new compound is the stable form of one of the structures
suggested. Since a condensation product has the fluorescent
color of DPNH it also should contain a quinone structure.

Having demonstrated a complete reversibility of the
glyoxylate inhibition of malic and lactic dehydrogenase by
excess DPN we then examined whether excess DPN could reverse
glyoxylate inhibition of mitochondria preparations. In the
experiment with malate as a substrate for mitochondrial
preparation, only partial reversibility (47%) of the
glyoxylate inhibitory effect was obtained by employing DPN
in excess (Figure 20).

Experiments with pure malic dehydrogenase were run

at pH 10.5 and with mitochondria preparations at pH 7.3 and

112

FIGURE 20

Effect of glyoxylate and DPN on malate oxidation

by green pea mitochondria.

1 - 0.001 M or 0.002 M DPN

2 - 0.001 M DPN and 40 ficmoles per flask of
glyoxylate

3 - 0.002 M DPN and 40 ptmoles per flask of
glyoxylate

Each flask contained 10 zemoles of malate. Final
pH was 7.3 and total volume of reactants 3.9 ml.
Assay conditions and mitochondria preparation was

as described in the Materials and Methods section.

 

 

,/”l 02 uptake

11}

 

 

FIGURE 20
1502—
X0
. ,//
120‘“ 1 ——9—-—-—/
/,
_/
/«'
901
1
1 1
60 L- 1 1 x __-.3
, -' : -//,/*
1 // y (Y
1 /
30 r- / ° 2
1 g/
1 C//l
’ e . -.:.-- o— - --~:------- 4)
/_* _W _ 1 n J 1_m__. -1
10 20 30 40 50 60

Time in minutes

the
DP};

1‘6‘

114

the higher pH would favor the condensation of glyoxylate with
DPNH (106, 107). Thus we could not expect as complete
reversibility by DPN of an inhibition with the complex
mitochondrial system. The failure of excess of DPN to re-
verse the glyoxylate inhibition of succinate oxidation by
mitochondria is consistent with the fact that succinic de-
hydrogenase requires FMN instead of DPN. Perhaps glyoxylate
also complexes with FMN which hypothesis could be checked

in a similar manner as done for glyoxylate condensation with

DPN.

SUMMARY

Glycolic acid oxidase is active in the cell-free ex-
tracts from green leaves but not in sheaths of leaves or in
etiolated leaves. The enzyme is predominantly located in
the cytoplasm although some enzymatic activity is associated
with mitochondria and chloroplast fractions.

An apoenzyme is present in sheaths or etiolated
leaves, and upon addition of FMN in excess to the homo-
genates of these tissues, an active enzyme is obtained.

Exposure of etiolated plants to the light results in
a several fold increase of active enzyme and of more apo-
enzyme. The increase in this enzyme is associated with
greening of the plant. This change may be in part explained
by the fact that green plants contain more FMN than etio-
lated plants. However, the content of FMN in types of
plants is 10'8 M which is below the 10"4 M concentration of
flavin necessary to activate the enzyme in 31339.

Enzyme can be activated in zitgg'by preincubation of
a cell-free sap in the cold with its substrates, glycolate,
lactate or ee-hydroxybutyrate. Ammonium sulfate precipitates
of this apoenzyme were not activated by incubation with
glycolate. The enzyme can also be activated ig‘zizg without

light by feeding etiolated plants glycolate, lactate,

116

aL-hydroxybutyrate or even glyoxylate.

Both glycolate and glyoxylate were shown to be ef-
fective inhibitors of the oxidation of citric acid cycle
intermediates by plant mitochondria. Glyoxylate was the
more potent inhibitor. Because glycolate was oxidized by
plant mitochondria by the associated glycolic acid oxidase
it was assumed that inhibitory effects of glycolate could be
ascribed to formation of glyoxylate. Glyoxylate also
severely inhibited isolated malic and lactic dehydrogenase.
Upon the addition of DPN in excess to these enzymes or to
mitochondria with malate as a substrate, the inhibitory effect
of glyoxylate was reversed. The inhibitory action of
glyoxylate was caused by a non-enzymatic formation of complex

with reduced DPNH. This complex was detected by paper

chromatography.

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CHEWSWY LIBRARY

 

 

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