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NUCLEOSIDE PHOSPHOTRANSFERASE,‘ Paosmomommx 1, ‘ ‘1 Q 7;-
AND cvcuc NUCLEOTIDE PHOSPHODlESTERASE : -
ACTMTIES OF CARROT LEAVES ' '

Theazis for the Degree of Ph. D. f
MECHIGAN STATE UNIVERSITY
RALPH JOSEPH VENERE ,
1970 '

 

  
        

0 L I B R A R Y
Michigan State
mversity

  

rre-s cc: q

This is to certify that the
thesis entitled

Nucleoside Phosphotransferase, Phosphomonoesterase and
chlic Nucleotide Phosphodiesterase
Avtivities of Carrot Leaves

presented by

Ralph Joseph Venere

has been accepted towards fulfillment
of the requirements for

_2h..D.._ degree in Wology

 

Major professor

Date ?»£..1¢,/7 7/

0-7639

 

ABSTRACT

NUCLEOSIDE PHOSPHOTRANSFERASE, PHOSPHOMONOESTERASE
AND CYCLIC NUCLEOTIDE PHOSPHODIESTERASE
ACTIVITIES OF CARROT LEAVES

BY

Ralph Joseph Venere

The objective of the research reported in the
first section of this two part thesis was to determine
whether carrot leaf phosphotransferase, an enzyme which
transfers phosphate from organic phosphate esters to
nucleosides, possesses inherent hydrolytic activity toward
a phosphate donor, p-nitrophenyl phosphate. The enzyme
was fractionated with acetone and ammonium sulfate and
purified further by successive ionic and pH gradient
elution from DEAE-cellulose columns. The elution profiles
of transferase and hydrolase activities showed two
separate bands that were congruent. The fractions con-
taining major transferase activity were pooled, dialyzed,
lyophilized and this purified preparation was used for
further investigations. Centrifugation of the preparation
in sucrose density gradients at pH 5.0 revealed the
presence of single overlapping, but not congruent, bands

for the two activities. Sedimentation at lower pH values

Ralph Joseph Venere

(4.5-3.0) or in 3 M urea demonstrated the presence of
several bands of transferase and several bands of
phosphatase activities, also overlapping but not con-
gruent. The profiles from Sephadex G-200 columns
indicated the presence of three species of phospho-
transferase having approximate molecular weights of
53,000, 100,000 and 200,000 which were congruent with
phosphatase activities. This suggests the existence of
monomeric, dimeric and tetrameric forms of the enzymes.
Atypical Michaelis plots were obtained for phosphotrans-
ferase at low concentrations of the donor but not at high
concentrations; a maximum Hill coefficient (n) of 2.3
was obtained when determinations were made with enzyme

treated with 0.05 M NaClO Ca salt favoring subunit

4:
dissociation. Transferase activity differed greatly from
phosphatase activity in sensitivity to heat, to denatur-
ants, to inhibitors and stimulators and to pH.

It is concluded that phosphotransferase possesses
two donor sites and is a protomer which probably possesseS'
hydrolytic activity. The data suggest however that
hydrolase and transferase sites are different and that
non-identical subunits may be derived from the parent
enzyme(s).

The objectives of the research reported in the

second section were two-fold. The first was to determine

the kinds of phosphate esters hydrolyzed by a crude carrot

Ralph Joseph Venere

leaf phosphotransferase preparation; the second was to
examine some properties of a system in this preparation
which hydrolyzed cyclic nucleotides to inorganic
phosphate and adenosine.

All of the phosphate esters tested were
hydrolyzed with the exception of Vitamin 812' Hence,
the crude preparation may be of some value in proving
the presence of phosphate in newly-discovered phosphate
compounds. Of particular interest was the fact that
3',5'-cyclic nucleotides were hydrolyzed to inorganic
phosphate. The hydrolysis of 3',5'-cyclic adenosine
monOphosphate was increased by Mg2+ (0.4 mM); Mn2+
(0.4 mM) and F- (6.6 mM) were inhibitory. The major
product of the phosphodiesterase component of the
system was 3'-adenosine monOphosphate, but small amounts
of the 5'-isomer were also detected. An apparent Km of
0.91 mM was determined for diesterase with 3',5'-cyc1ic
adenosine monOphosphate and an apparent Km of 3.33 mM
was calculated for nucleotidase activity with 3'-
adenosine monOphosphate. No apparent substrate speci-
ficity for the system was noted since both purine and
pyrimidine 3',5'-cyclic nucleotides were hydrolyzed to about
the same extent. The preparation also hydrolyzed 2',3'-
cyclic adenosine monOphosphate; an apparent Km of 1.45
mM.was calculated for this substrate. Centrifugation in

sucrose gradients yielded three bands hydrolyzing

Ralph Joseph Venere

2',3'-cyclic adenosine monOphosphate but only two
hydrolyzing 3',5'-cyclic adenosine monOphosphate.

It is concluded that higher plants do indeed
possess enzymes capable of hydrolyzing 3',5'-cyclic
nucleotides. This suggests that such compounds may be
physiologically active in higher plant tissue. The
prOperties described for the system indicate that the
phosphodiesterases have different properties than those

described from animal sources.

NUCLEOSIDE PHOSPHOTRANSFERASE, PHOSPHOMONOESTERASE
AND CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

ACTIVITIES OF CARROT LEAVES

By

Ralph Joseph Venere

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1970

To my wife, Florence
and son, Ralph

ii

ACKNOWLEDGMENT S

It is extremely difficult to acknowledge everyone
who has made a contribution towards this study. It is
even more difficult to express the gratitude that must be
extended to Dr. Clifford J. Pollard, my major professor.
Only through his intense desire to provide everyone of
his students with all the guidance and encouragement that
he is able to provide could this study have been completed.
The perseverance and dedication to his work which he
always displayed provided much needed inspiration. Sincere
appreciation must also be extended Us Drs. N. E. Good,
R. P. Scheffer and F. M. Rottman who served as members of
the guidance committee. A very special thank you must be
given to Dr. Jessica Reimann for her instruction and
advice during the kinetic studies. I would also like to
thank Dr. Geoffrey Kennedy for the technical assistance
which he provided concerning the preparation of the
Sephadex column.

This work was supported in part by a Biomedical
Sciences Support Grant from the National Institutes of

Health .

iii

TABLE OF CONTENTS

Page
DEDICATION . . . . . . . . . . . . . . ii
ACKNOWLEDGMENTS . . . . . . . . . . . . . iii
LIST OF TABLES . . . . . . . . . . . . . Vi

LIST OF FIGURES . . . . . . . . . . . . . Vii

SECTION I. NUCLEOSIDE PHOSPHOTRANSFERASE
AND PHOSPHOMONOESTERASE

INTRODUCTION . . . . . . . . . . . . . . 2
MATERIALS AND METHODS . . . . . . . . . . . 7
Plant Source . . . . . . . . . . . . . 7
Chemicals . . . . . . . . . . . . . . 7
Enzyme Preparations . . . . . . . . . . . 8
Protein Determinations . . . . . . . . . . 10
Enzyme Assays . . . . . . . . . . . . 11
Preparation and Assay of Sucrose Density Gradients. 13
Preparation of the Sephadex G-200 Column . . . . 15
RESULTS . . . . . . . . . . . . . . . 17
Modification of DEAE— Cellulose Column Chromatography
Purification Steps . . . . . . . . 17
A Compsrison of Some PrOperties of the Phospho-
transferase and Phosphomonoesterase Activities . 21
Time Course . . . . . . . . . . . . . 21
Effect of pH . . . . . . . . . . 22
Effect of Exposure to Acetate Buffer, pH 5.2 and
to Tris Buffer, pH 8.0 . . . . . . . . 24
Effect of Increasing Temperature . . . . . . 28
Effects of Monovalent and Divalent Cations . . 31
Inhibition by EDTA and NaF . . . . . . . . 33
Effect of Denaturing Agents . . . . . . . 35
Kinetic Studies on Phosphotransferase and
PhOSphomonoesterase Activities . . . . . . 42
Sedimentation in Sucrose Density Gradients . . 52
Evidence that Phosphotransferase Possesses
Hydrolytic Activity . . . . . . . . . . 59

iv

Page

DISCUSSION 0 O O O O O O O O C O O O O 66

SECTION II. CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

INTRODUCTION 0 O O O O I I O O I O I O O 7 6
MATERIALS AND METHODS . . . . . . . . . . . 80
Enzyme Source . . . . . . . . . . . . . 80
Chemicals I O O O O O O O I O O O O O 80
Enzyme Assays . . . . . . . . . . 80
Determination of Nucleotide Intermediate . . . . 82
Sucrose Density Gradients . . . . . . . . . 83
RESULTS 0 O O O O I O O O O O O O O O 85
Substrate Specificity of Crude Phosphotransferase . 85
Some Properties of the Cyclic Nucleotide
Phosphodiesterase System from Carrot Leaves . . 87
Time Course . . . . . . . . . . . . . 87
Effect of Temperature . . . . . . . . . 87
pH Optimum . . . . . . . . . 91
Effect of MgZ+, Mn2+ and F . . . . . . . 91
Identification of Reaction Products . . . . . 94
Sucrose Density Gradient Analysis . . . . . 95
Michaelis Constants . . . . . . . . . . 95
DISCUSSION 0 I O O O O O O O O O O O O 99

LITERATURE CITED 0 O O O O O O O C C O O 103

LIST OF TABLES

Effect of time of incubation on the
phosphorylation ratio . . . . . . . .

Effect of exposure to high temperatures on
enzymic activity . . . . . . . . . .

Effect of cytidine on the activity of phospho-
transferase exposed at 60°C . . . . . .

Effect of various cations on enzymic activity .

Approximate molecular weight of three phospho-
transferase bands eluted from a Sephadex
G-200 COlumn o o o o o o o o o o 0

Relative apparent rates of hydrolysis of
various organic phosphate esters by "Crude
Phosphotransferase" from carrot leaves . .

Relative apparent rates of hydrolysis of various
cyclic nucleotide substrates by "Crude
Phosphotransferase" from carrot leaves . .

Effect of Mgz+, Mn2+, and F- on phosphodiester-
ase activity . . . . . . . . . . .

vi

Page

22

30

32

34

63

86

88

93

 

lik‘va.’ urn I.J¢

10.

ll.

12.

13.

LIST OF FIGURES

Sodium chloride gradient elution of "Crude
Phosphotransferase" from a DEAE-cellulose
collumn . . . . . . . . . . . .

pH gradient elution of "Partially Purified
Phosphotransferase" from a DEAE—cellulose
column . . . . . . . . . . . . .

Time courses of phosphotransferase and
phosphomonoesterase activities . . . . .

Effect of pH on enzyme activity . . . . .

Effect of exposure to acetate or tris buffers
on enzyme activity . . . . . . . . .

Effect of increasing temperature on enzyme
aCtiVitieS O O O O O O O O O O 0

Effect of EDTA concentration on enzyme
activity . . . . . . . . . . . .

Effect of sodium fluoride on enzyme activity .

Effect of HgClz concentration on phospho-
transferase and phosphomonoesterase
activities . . . . . . . . . . .

Activation and inactivation of enzyme activity
by urea . . . . . . . . . . . .

Effects of alcohols and acetone on enzyme
activity . . . . . . . . . . . .

Effect of increasing amounts of phosphate
donor substrate on the velocity of phospho-
transferase . . . . . . . . . . .

'Velocity of phosphotransferase as a function of

phosphate donor concentration obtained for
acetate and perchlorate treated samples . .

vii

Page

18

20

23
25

27

29

36

37

39

4O

41

43

47

Figure Page

14. Double reciprocal plot for pNPP in the
phosphotransferase reaction . . . . . . 48

15. Hill plots of the phosphotransferase reaction. 50

16. Reciprocal plots of phosphotransferase at high
pNPP concentrations . . . . . . . . 51

1?. Kinetic pattern of pNPP saturation in the
phOSphomonoesterase reaction . . . . . 53

18. Reciprocal plot for pNPP in the phosphomono-
esterase reaction . . . . . . . . . 54

19. Ultracentrifugation pattern of enzyme activi-
ties as a function of pH . . . . . . . 56

20. Effect of urea on the sedimentation profiles
of phosphotransferase and phosphomono-
esterase . . . . . . . . . . . . 58

21. Chromatography of "Purified Phosphotransferase"
on a Sephadex G-200 column . . . . . . 61

22. Determination of molecular weights of
phosphotransferase by chromatography on
Sephadex G-200 . . . . . . . . . . 62

23. Ultracentrifugation pattern of "Purified
Phosphotransferase" in sucrose gradients at
pH 5.2 in 0.1 M acetate . . . . . . . 64

24. Time course of the formation of Pi by the
crude phosphodiesterase preparation . . . 89

25. Phosphodiesterase activity as a function of
increasing temperature . . . . . . . 90

26. Effect of pH on phosphodiesterase activity . 92
27. Ultracentrifugation patterns of phosphodi-

esterase activity with 3'c5'-cAMP and 2',3'-

cAMP as substrate . . . . . . . . . 96
28. Comparison of the Michaelis constants for 3'-

nucleotidase and cyclic nucleotide phosphodi-
esterase activities . . . . . . . . 97

viii

SECTION I

NUCLEOSIDE PHOSPHOTRANSFERASE AND

PHOSPHOMONOESTERASE

INTRODUCTION

The first reports concerning enzymes capable of
transferring organically esterified phosphate to nucleo-
side acceptors were made by Brawerman and Chargaff (1, 2)
in 1953. Subsequent studies (3, 4) provided details with
respect to specificities of donors and acceptors, as well
as a determination of the nucleotide isomer formed.
Information was also given on the distribution of the
«enzymes in different cells and tissues. As a result of
‘these studies a provisional classification of the various
leinds of enzymes found was provided (5). Thus, the enzymes
from plants and some microorganisms preferred a 5'-nucleo-
'tide donor and produced only 5'-nucleotides as products.
Ihizymes from most mammalian tissue preferred phenyl
{fluDSphate as donor; giving rise to 5'-nucleotides as well
as snail quantities (5-10%) of the 2'-and 3'-isomers.
Human.prostate preparations, however, utilized phenyl
PhOSphate or 3'-nuc1eotide equally as donors, preferred
deoxyriboside acceptors and produced, in addition to the
5"ntu:1eotides, a significant amount of 3'-nucleotides.
When Jribosides were used as acceptors considerable amounts
0f tins 2'-nuc1eotides were formed. These enzymes were

calls“: nucleoside phosphotransferases due to their

specificity for nucleoside acceptors. The pH optimum of
5.2 in acetate buffer was the same as the pH Optimum for
the acid phosphomonoesterase activity that was present in
all of the preparations tested.

Several years later Katagiri g£_§1. (6), utilizing
a cell free extract as well as a partially purified enzyme
preparation from Escherichia coli, showed that a predomi-
nance of 3'-inosinic acid was formed when p-nitrOphenyl-
phosphate (pNPP) served as donor and inosine as the
acceptor in the phosphotransferase reaction. Mitsugi
g£_§l, (7) and Mitsugi (8, 9) studied in detail the dis-
tribution and prOperties of phosphotransferases in a large
number of cell-free extracts of bacterial origin. In a
series of papers in which the acceptor specificity of
nucleoside phosphotransferase was studied (10-15), it was
concluded that the isomeric product formed was dependent
on the particular strain of bacteria used as the enzyme
source. Thus, they did not consider the classification
set forth by Brawerman and Chargaff to be absolute. It
should be mentioned that Roberts (16) also took exception
'to the classification of Brawerman and Chargaff. He
<3laimed the separation of a distinct nucleoside phospho-
transferase enzyme from an Azgna_leaf preparation that was
able to synthesize adenosine 3'-phosphate from adenosine 5'-
phosphate and adenosine. This was in contrast to the carrot

root preparation which could form only the 5'-isomer.

 

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The presence of these transfer enzymes in animal,
bacterial and plant cells provides some support for the
possibility that nucleoside phosphotransferases take part
in the cellular biosynthesis of nucleotides. Additional
support for this role is suggested from investigations
concerning the activity of these enzymes in relation to
the physiological state of the tissue being studied.
Brawerman and Chargaff (4) showed that a significant
increase in phosphotransferase activity occurred during
the germination of wheat, the regeneration of rabbit
muscle, and during the growth of bacterial cells. This
increase in phosphotransferase activity was shown to far
surpass the increase in phosphomonoesterase activity.
Moreover, during the germination of wheat the increase of
phosphomonoesterase activity was found in both the endo-
sperm and in the shoot whereas the rise in phosphotrans-
ferase activity was found only in the growing shoot.

The presence of phOSphomonoesterase activity in
all preparations used in the above studies tended to
obscure the true synthetic capacity of the phosphotrans-
ferase. The importance of the enzyme in nucleotide
synthesis could be evaluated better if it could be
ascertained whether or not an enzyme which is free from
hydrolytic activity exists. Tunis and Chargaff (l7),
utilizing an enzyme preparation from carrot root tissue,

attempted.to separate proteins responsible for the two

activities. Although unsuccessful, they were able to
demonstrate that c0pper or zinc ions specifically inhibited
the phosphomonoesterase aCtivity. Further treatment of
their preparation with Celite removed additional hydrolytic
activity; it has also been reported that the enzymes can

be separated by paper electrophoresis (18). It was con-
cluded that two different enzymes were responsible for the
two observed activities. Mitsugi (9) also claimed that
the bacterial nucleoside phosphotransferases were distinct
from the acid phosphomonoesterases on the basis of the
(iifferential effects of various inhibitors and metallic
ions on both activities.

Brunngraber and Chargaff (19) prepared carrot root
jphosphotransferase by a method affording a purification of
Inore than 1700 fold. Nevertheless, a significant amount of
hydrolytic activity was present in the preparation. It
teas not determined whether the phosphomonoesterase activity
\vas associated with the phosphotransferase enzyme or
\vhether it was merely present as a contaminant of the
preparation.

Recently, Becker and Pollard (20) devised a purifi-
<:ation scheme for the nucleoside phOSphotransferases of
carrot leaves. This preparation also contained a significant
amount of phOSphomonoesterase activity. No attempts were
made to determine conclusively if the phosphotransferase

activity was associated with a protein distinct from the

phosphomonoesterase. However, it was mentioned that
distinct entities might exist; on the basis of slight
differences observed in the elution profiles from the
diethylaminoethyl (DEAE)-cellulose purification steps (21).

The objective of this section of the thesis was to
investigate more fully the nucleoside phosphotransferase
of carrot leaves in an attempt to discern whether the

enzyme possesses phOSphomonoesterase activity.

MATERIALS AND METHODS

Plant Source
Seeds of Burpee's Golden Chantenay carrot (Daucus
carota) variety were purchased from a local supplier. The
seeds were sowed by hand in the field in late June and the
plants were harvested three months afterwards. Healthy
green leaves, free from visible signs of infection, were
cut from the large taproot, washed thoroughly in tap water

and utilized as a source for the enzyme preparations.

Chemicals

 

All chemicals were purchased commercially. They
were of the highest purity available and were not subjected
to further purification. Tritiated uridine (ICN, Sp. Act:
616 mc/mM), kindly donated by Larry Pelcher, was used as
acceptor in several studies. The radiochemical purity of
this material was found to be greater than 98% by chroma-
tography of the sample in a system of 75% ethanol--10 per
cent saturated (NH4)ZSO4 impregnated paper (22). DEAE-
Cellulose was purchased from Sigma Chemical Company, St.
Louis, Missouri. Sephadex G-200 was kindly donated by

Dr. Fritz Rottman.

Enzyme Preparations

 

Phosphotransferase was prepared by a method very
similar to that described by Becker and Pollard (20).
All operations were performedjxithe cold (3-4°C). Two
kilograms of the washed leaves were homogenized in a
Waring blendor with one liter of 0.1 M sodium acetate at
pH 5.1 by successive re-homogenizations and filtrations
through cheesecloth. The leaves were added in 50-100
gram portions to about 300 m1 of buffer, homogenized and
the slurry was filtered through the cheesecloth into a
stainless steel beaker set in an ice bath. This procedure
was continued until all the leaves were homogenized.

Initial purification was made by collecting the
fraction precipitating between 30 and 70 per cent by volume
of cold acetone (-20°C) by centrifugation (10,000 X g for
10 minutes). The pellet was dissolved in cold distilled
'water and further purified by.(NH4)ZSO4 fractionation.
The fraction precipitating between 40 and 80 per cent of
saturation was collected by centrifugation (10,000 X g for
10 minutes) and dissolved in a minimum amount of 0.1 M
acetate buffer of pH 5.0. This solution was subsequently
dialyzed against this same buffer, lyOphilized and stored
at -20°C for further use. This preparation was designated
"Crude Phosphotransferase."

For further purification, the "Crude Phosphotrans-

ferase" preparation was applied to a 22 X 1.5 cm column of

diethylaminoethyl (DEAE)-ce11ulose (acid and base washed)
that had been equilibrated with 500 m1 of a buffer con—
sisting of 0.005 M MgAc and 0.01 M Tris (MAT-buffer),
pH 7.5. The protein was eluted from the column with a
linear NaCl gradient (0-1 M) prepared according to the
principles of the double chambered gradient preparing
device of Britten and Roberts (23). The device employed
in the present studies, however, consisted of two plastic
bottles connected to the column in series with small
diameter rubber tubing rather than the doubled chambered
block of Lucite described by these authors. The bottle
connected directly to the column served as the mixing
chamber. Solutions in this chamber were mixed with a
magnetic stirring bar. The second bottle (reservoir
chamber) contained the buffered NaCl solution.

The flow rate in all experiments was maintained at
about 24 ml per hour. The eluant was collected in 10 ml
fractions with a fraction collector. Aliquots were assayed
for the desired enzymatic activity and those fractions
containing peak transferase activities were pooled,
dialyzed overnight against four liters of cold distilled
water and concentrated by freeze-drying. This preparation,
designated "Partially Purified Phosphotransferase," was
stored at -20°C until needed.

To obtain phosphotransferase of a higher degree of

purity, a second DEAE-cellulose column was prepared as

10

described above except that it was equilibrated overnight
with 500 ml of a buffer consisting of 0.05 M malic acid,
0.025 M acetic acid and 0.05 M maleic acid adjusted to pH
6.0 with NaOH (MAM-buffer). "Partially Purified Phospho-
transferase" preparation (100 mg of protein) was taken

up in cold glass distilled water, dialyzed against MAM-
buffer of pH 6.0 and subsequently applied to the DEAE-
cellulose column. Elution was carried out with a linear
pH gradient (1000 ml) prepared with the same device
described above. Thus, 500 m1 of MAM—buffer (pH 6.0) was
placed in the mixing chamber and 500 ml of MAM-buffer

(pH 1.2) was added to the reservoir chamber and the eluate
collected in 10 ml fractions. Fractions exhibiting peak
transferase activity were pooled and dialyzed overnight
against two liters of cold glass distilled water. The
dialyzed protein was lyOphilized and subsequently stored
at -20°C. This preparation was designated "Purified

Phosphotransferase."

‘ Protein Determinations

 

Proteins eluting from the DEAE-cellulose columns
were estimated by their absorbance at 280 nm. When the
Protein concentration was too low to be measured adequately
by this means, the method described by Lowry §E_al. (24)
was used. Protein concentration of the various enzyme

Preparations was determined by the method described by

ll

Warburg and Christian (25). It should be noted that the
protein content in most cases where direct SpectrOphoto-
metric assays were made is merely an approximation of the
true amount present. The presence of a brown color,
probably due to polyphenolic material, was observed in
practically every preparation. This color no doubt inter-
fered with the determinations. The only material free
from this brown color was that obtained after passage
through the second DEAE-cellulose column and that eluted

from a Sephadex G-200 column.

Enzyme Assays

 

Nucleoside Phosphotransferase.--The standard
assay used for the determination of phosphotransferase
activity consisted of incubating 3 Umoles of pNPP, lu
Inole of cytidine, and the appropriate amount of enzyme
in a final volume of 0.3 ml of 0.1 M sodium acetate
buffer of pH 5.0. The enzyme solution had been diluted
so as to insure linear product formation during incubation.
The incubation time was usually one-half to one hour at
37°C and the reaction was stopped by boiling for 5
Ininutes in a water bath.

The amount of enzymically formed nucleotide was
(determined by separation of the entire reaction mixture
\Iia paper electrophoresis. The electrolyte consisted of
0.01 M EDTA (tetrasodium salt) to which 0.34 ml of

Pyridine per liter was added. The solution was adjusted

12

to pH 3.5 with glacial acetic acid. Electrophoresis was
of 6 hours duration at 400 volts potential (13.3 volts/cm).

ElectrOphoresis was followed by elution of the UV
absorbing area of the dried electrophoretograms correspond-
ing to cytidine monOphosphate (CMP) with 0.1 N HCl and
spectrOphotometric analysis at 280 nm. Readings were made
against a control sample treated identically except that
enzyme was replaced by an equal volume of buffer or by
enzyme boiled at 100°C for five minutes.

When 3H-uridine (6 mM) was used as the phosphate
acceptor, experiments were conducted in a manner similar
to that described above. After the reaction mixture was
streaked onto the electrOphoresis paper, unlabelled
carrier uridine monOphosphate (UMP) was applied. Electro-
phoresis was performed as given above and the UV light
absorbing area was cut from each paper strip in 2 cm
sections. These were placed in counting vials containing
15 m1 of toluene in which the phosphors 1,4—bis-2-(4-
methyl-S-phenyloxazolyl)-benzene (0.3 g/liter) and
2,5 diphenyloxazole (5 g/liter) were dissolved. Radio-
activity of samples was determined in a Packard 3003 Tri-
Carb scintillation spectrometer. Controls, using
3H-uridine but no active enzyme were also run. Trans~
ferase activity is expressed either as mumoles of
nucleotide formed per hour or as cmp incorporated into

3H-UMP per hour.

mob
en:
InCI
pro<
Act:
p-n:
mini
(27)
cont
agai

eXpr

of p
is t
Phos
lar .

of t]

with
by Ma
2'3 m.

in the

l3

Phosphomonoesterase.--For the standard assay, 3p

 

moles of organic phosphate ester were incubated with the
enzyme in 0.3 ml of 0.10 M sodium acetate of pH 5.0.
Incubations were carried out over a period during which
product formation was linear; usually 10 to 30 minutes.
Activity was estimated by determining the amount of
p-nitr0phenol (pNP) released from pNPP (26) or by deter-
mining the amount of inorganic phosphate (Pi) released
(27). ApprOpriate substrate controls and boiled enzyme
controls were utilized. Experimental samples were read
against these controls. Phosphomonoesterase activity is

expressed as mumoles of pNP released per hour.

The phosphorylation ration (#) is the molar ratio
of phosphate transferred to p-nitrophenol released. R/R°
is the ratio of the rate of phosphotransferase or
phosphomonoesterase activity in the presence of a particu-
1ar compound to the rate of either activity in the absence
of the compound.

Preparation and Assay of Sucrose
Density Gradients

Linear sucrose gradients (5%-20%) were prepared
with a double chambered device similar to that described
by Martin and Ames (28). A solution of 20% sucrose (2.2-
2.3 ml) in 0.1 M acetate buffer (pH 3-5.2) was contained

.in the mixing chamber and an equal volume of a 5% sucrose

 

solut:
The S'
the t1
an ai
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milli.
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four .
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PhOSp1
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deterr

14

solution in the same buffer was placed in the reservoir.
The stopcock connecting the two chambers was opened and
the two solutions were mixed by a glass rod attached to
an air driven stirrer. In certain instances urea (3M)
was added to the buffered sucrose Solutions prior to mix-
ing. The prepared gradients were chilled at 4°C for one
hour at which time a solution containing 0.12 to 3.0
milligrams of protein was very carefully pipetted onto the
gradient surface. The gradients were sealed with 0.15-
0.20 ml of paraffin oil and then spun in a SW 39L rotor
at 38,000 rpm for 31-34 hours at l-2°C.

Fractions were collected by piercing the tubes
at the bottom with a needle device set up in conjunction
with a drop counting device which was preset to collect
four drOpS per fraction. Usually, 30-32 fractions were
collected (approximately 0.155 ml/fraction). When
gradients were prepared in acetate buffer at pH 3.0 or
in the presence of 3M urea, a noticeable increase in the
number of fractions was detected (42-44 fractions).
Apparently, this was due to a reduction in the drop size.

Each fraction was diluted with 1 ml of 0.1 M
acetate buffer of pH 5.0. Aliquots of 0.10 ml were with-
drawn and assayed for enzymatic activity. Assays for
phosphotransferase were carried out with a donor:acceptor
ratio of 5:1. The phosphomonoesterase activity was

determined with 5 umoles of pNPP. The amount of protein

 

pres

meth

110'
then
atel}
5.2
The c
month
trans
pH 5
aceta
of th

fract

15

present was too low to be detected by commonly employed

methods.

Preparation of the Sephadex G-200 Column

 

Sephadex G-200 was heated in water at 100°C briefly,
allowed to cool and kept at 4°C overnight. The slurry was
then used to prepare a column (45 X 2 cm) which was immedi-
ately equilibrated with 0.1 M sodium acetate buffer of pH
5.2 (200 ml) at a flow rate of approximately 10 ml per hour.
The column was then left in the cold room at 4°C for two
months after which time a solution of "Purified Phospho-
transferase" (14 mg of protein in 2 ml of 0.1 M acetate,
pH 5.2) was applied and subsequently eluted with the same
acetate buffer and at the flow rate used for equilibration
of the column. Fractions of 3.8 ml were collected with a
fraction collector.

The column was calibrated by determining the elution
volume (Ve) of reference standards. The void volume (Vo)
was determined with blue dextran. Reference standards of
cytochrome c and hemoglobin were detected spectrOphotometri-
cally at 412 nm, apoferritin at 230 nm and blue dextran
at 625 nm. Catalase was assayed by following the decrease
in absorbance at 240 nm due to the destruction of hydrogen
peroxide, as described by Chance and Maehly (29), and
alkaline phosphatase was detected by the increase in
absorbance of pNP at 410 nm in 0.05 M HEPES at pH 8.5.

A plot of elution volume against the log molecular weight

of the reference standards was prepared and approximate

16

molecular weights of phosphotransferase and phosphomono-

esterase were determined from this plot.

RESULTS

Modification of DEAE-Cellulose Column
Chromatography Purification Steps

In the purification scheme for the preparation of
nucleoside phosphotransferase enzyme from carrot leaves,
two DEAE-cellulose chromatography steps were employed
by Becker and Pollard (20). A linear gradient of
buffered sodium chloride was used for the elution of
protein from the first column and the protein applied to a
second column was eluted with a pH gradient.

It was thought that successful separation of the
phosphotransferase and phosphomonoesterase activities might
be achieved by modifying their elution conditions. Thus,
changes were made in eluant volume, sodium chloride con-
centration and steepness of the pH gradient in an effort to
separate the activities. Only the final conditions used
to obtain the "Purified Phosphotransferase" will be
described.

The conditions utilized for the elution of "Crude
Phosphotransferase" (200 mg protein) by a linear gradient
of buffered sodium chloride were similar to those described
by Becker and Pollard (20) except for the eluant volume.

Figure 1 is a typical elution profile for phosphotransferase

l7

18

v

/
S
4
|_a
0
ON

mu moles pNP/hr x 10—2
W
a

mu moles CMP/hr x 10 2
H
is

     

 

 

 

 

 

RWWW
///O /

 

 

4b 5'0 '6'0 7‘6 "86' £57) 100

LA)
0

Fraction Number

Figure 1. Sodium chloride gradient elution of "Crude Phosphoa

Protein (OD 280)

transferase from a DEAE-cellulose column. A column (22 x 1.5 cm) was

prepared and equilibrated with 0.01 M tris containing 0.005 M
MgAc buffered at pH 7.5. The column was developed with one liter
of a linear NaCl gradient (0-1 M) prepared in the same buffer.

The flow rate was maintained at 24 ml per hour and the volume of
the fractions collected was 10 ml. Indicated in the figure are
the elution profiles obtained for phosphomonoesterase (C>),
phosphotransferase ([3) and protein (--).‘ The hatched area
indicates the fractions that were pooled for further purification.
This preparation is "Partially Purified Phosphotransferase".

 

activ:

the v

ester
capat
Phos;
that

trans
trans

respc

Prote
linea
PH 1.
used
Steer
3 vex
be a1

00th

the E
two i

of h}

h:
‘*QUr

PhOSp

19

activity, phosphomonoesterase activity and protein when
the volume of eluant used was one liter.

The broad elution profile for the phosphomono-
esterase activity suggested that more than one enzyme
capable of hydrolyzing pNPP was present in the "Crude
Phosphotransferase" preparation. It was possible, then,
that one of these enzymes was also responsible for the
transferase activity. However, the possibility that a
transferase enzyme which was devoid of phosphatase was
responsible for this activity could not be ruled out.

"Partially Purified PhOSphotransferase" (100 mg
protein) was eluted from a DEAE-cellulose column with a
linear pH gradient (1000 m1) buffered from pH 6.0 to
pH 1.2 instead of the range of from pH 5.75 to pH 1.8
used by Becker and Pollard (20). This change in the
steepness of the pH gradient was made because in so doing
a very active band of phosphomonoesterase activity could
be almost completely removed from the phosphotransferase
activity (Figure 2).

It should also be noted that under these conditions
the phosphotransferase activity eluted from the column in
two bands. Both transferase bands were eluted with bands
of hydrolytic activity.

Fractions indicated by the hatched areas in
Figure 2A were pooled and served as the "Purified

Phosphotransferase" preparation.

20

.muaon mo oocuofi ecu mo cocaaumumo mo caououm How waflmoum cofiucam .m
.smmmummmamuuonmmonm.ooflmwncm= ma coaumnmmmnm mcHuHSmmH one .cmaoom mcowuomum

on“ mouooaocw mono venous: one _.Anuv ommummmcmuuonmmosm can Anus ommumummocoaoammosm

mom maamoum coausam .d .HE ca mos mcowuooum ecu mo oEsHo> may one noon you He am on Macs
was open 30Hm.msa .Auao>ummmu may cw. N.H no mo Mommas mo HE com com Honsoso mcHNHE,mnu ca
Ao.m may Hmmmco mEMm wasp m0 H8 com zuw3 cwnmflanmumm me? cacaoo mop moam>mc on com: Anus
ucwficoum mm one .o.m mm on pace OHonE z mo.o one cfioo capoom z mmo.o .ofloo oaama z mo.o
mo mcflumflmcoo Hmmmsn m sua3.omumunwaflcwm mm3 Aao m.H x was cacaoo one .cacaoc omoasaawo
Imdmo o Eonm =mmouommcmnuocmmonm coflmansm maaowpuom= mo coauoao ucoflcoum mm .m madman

 

 

     

 

  

 

 

 

 

 

Honssz coauoenm noossz cowuoonm
om ov om om 0H OCH om om on om cm 0% on om 0H 0
ll IIIWI-rm / i - . - .. urns" 1. o ‘
Ju/fl \- H
7 e
To, V“ a
, a :N
1 m
d d .m
I x 1 n
w «.0- -1 we ..m m
a r I o
r. a .L
n s m
w, . m : a a
_L m o. wums C v Anna
S I u.
0 1
x
«.91 e M
1 m .
7v
m d [MW/AV :o

 

 

 

 

21

A Comparison of Some Properties of the
Phosphotransferase and Phosphomono-
esterase Activities

 

 

 

Since all attempts at separating transferase
activity from phosphomonoesterase activity by ion exchange
on DEAR-cellulose columns failed, it appeared likely that
both activities were associated with the same enzyme; it
would seem to be more than fortuitous that each transferase
eluted by a linear pH gradient was accompanied by a band
of hydrolytic activity (Figure 2). However, the possi-
bility also existed that the two activities were the
result of different enzymes. If the first case were true,
then an investigation of some general biochemical and
physical prOperties associated with bothactivities would
possibly yield similar results. However, if the two
activities were due to different enzymes, then marked dif—
ferences in preperties might be obtained. Thus, experiments
comparing some general prOperties of both activities were

conducted.

Time Course

In order to obtain significant results from these
investigations, it was necessary to make certain that
significant transfer of phosphate to the nucleoside
acceptor took place, that the release of hydrolysis
product was also measurable and that both were prOportional

to the time of incubation.

22

A time course of product formation for each of the
activities was determined (Figure 3). It can be seen that
the formation of the nucleotide and the hydrolysis of the
phosphate donor remained essentially linear when tested
under the standard assay conditions (molar ratio of donor:
acceptor--3:1). At the maximum time allowed for the
reaction (1 hr), 9.7% of the pNPP was hydrolyzed while 5.4%
of the cytidine was phosphorylated. Table 1 shows that the
phosphorylation ratio (phosphate transferred/pNP released)
declined at 1 hour of incubation. For this reason, an
incubation period of 30 minutes was normally used in most
studies. When it was noted that the initial rate of
hydrolytic activity was low the time of incubation was

extended accordingly.

TABLE l.--Effect of time of incubation on the phosphoryla-
tion ratio.

 

 

Time of Incubation (minutes) Phosphorylation Ratio
20 .208
40 .218
60 .185

 

Effect of pH

The pH optimum for the phosphotransferase reaction

in 0.1 M acetate buffer was found to be about pH 4.5

23

 

 

 

3.-
o
‘7'
3 -~ 50
H 2T T 40 0.
Q 5
e ° a.
m. u~30 3
O
3 51 E
1.. ..
2 2° 2
:L
s 8
o
o
10 20 30 40 so 60

Time (minutes)

Figure 3.
phosphomonoesterase activities.

Time courSes of phosphotransferase and

Phosphotransferase

is expressed as mu moles CMP synthesized per hour
(C1) and phosphomonoesterase as mu moles pNP

liberated per hour (C)).

 

_ _._____

(Fig
5.21

by B

phos;
furtl
seen
acti‘

compe

ester

24

(Ffiigure 4). This is somewhat less than the value of pH
5..2 determined by Becker (21) and the pH of 5.0 reported
by Brunngraber and Chargaff (16) .

Although a pH Optimum of 4.5 was noted for the
jpruosphotransferase reaction, it was decided to conduct
fiirther investigations at a pH of 5.0 since, as will be
seeen.later, at this pH the transferase and hydrolytic
arztivities sedimented in sucrose gradients as one band in
<2cunparison to the several bands detected at pH 4.5.

Two pH Optima were observed for the phosphomono-
esterase activity. The first appeared in the vicinity of
pfli 4.2; the second was close to pH 5.0. Brunngraber and
CHmargaff also reported two pH optima for the hydrolytic
euztivity present in their preparation. However they
curtained values of pH 5.0 and pH 6.0 to 6.5. No comparison
deth Becker's preparation could be made since no value for
hywirolytic activity was presented. It is possible that
digfferences in the purification methods account for the
VEtriations in pH optima noted for the various preparations.
Eifect of Exposure to Acetate

£2581)ng 5.2 and to Tris
.Efljfer, pH 8.0

 

Since the two activities exhibited differences in
PH Optima, it was thought that some information on the two
aetivities could be obtained by exposing the "Purified

Preparation" to different buffers whose pK values were on

25

31 «#30
N

I

O

" :3
>4 \
H 94
fl 2 1-20 5
3 e
"‘ '3
2’} s
v-1

0 I1
e 1- -—10 E
1

s

 

 

 

 

Figure 4. Effect of pH on enzyme activity.
(0) phosphotransferase; (O) phosphomonoesterase.

 

_.—-——

the
wer
kep
or

int

late
ace‘
tie:
that
dec]
Note
shor
retr
two-

aCti

uPOn
deal
buff
duIi:
the .
aflier
trans

a‘Ctir

26

the acid or alkaline side of neutrality. Thus, experiments

were conducted in which "Purified Phosphotransferase" was
kept either at pH 5.2 in 0.1 M acetate buffer (pK = 4.76)
or at pH 8.0 in 0.1 M Tris buffer (pK = 8.1). At
intervals aliquots were withdrawn and assayed.

Figure 5A Shows that both activities were stimu-
lated during the first fifteen minutes of exposure to
acetate buffer at pH 5.2. Although the per cent stimula-
tions are different, the curves resemble each other in
that the initial increase of activity is followed by a
decline in both transferase and hydrolytic activities.
Note that the effect on phosphomonoesterase is small and
short lived; after 60 minutes exposure the activity had
returned to the original level. On the other hand a
two-fold stimulation was noted in phosphotransferase
activity at that time.

In contrast to the stimulation of the activities
upon exposure to acetate buffer pH 5.2, both activities
declined when the preparation was kept at pH 8.0 in Tris
buffer (Figure 5B). The rate of loss was very similar
during the first ten minutes of exposure. Afterwards,
the transferase activity declined at a faster rate. Thus,
after 60 minutes of exposure only 2% of the original
transferase activity remained whereas 48% of the hydrolytic

activity was retained.

% Initial Activity

 

27

 

 

 

 

l A l n l n l L
‘ U V V f v ‘ I r f

10 20 30 40 50 60 0 10 20 30 40 50 60

Time (Minutes) Time (Minutes)

Figure 5. Effect of exposure to acetate or tris buffers on-
enzyme activity. Individual 0.01 ml portiOns of "Purified
Phosphotransferase" were diluted with 9.99 ml of either (A)
0.1 M acetate (pH 5.2) or (B) 0.1 M tris (pH 8.0). The
enzyme solutions were kept at 23°C. At the indicated time
intervals, 0.1 ml aliquots of the enzyme solution were with-
drawn and assayed for either phosphotransferase ([3) or
phosphomonoesterase (CD); The curves presented in the figure
represent the values obtained from one of three separate
experiments.

28

Effect of Increasing Temperature

 

Like the majority of chemical reactions, enzymat-
ically controlled reactions show increased rates at
increased temperatures. However, under a given set of
conditions, there is a temperature beyond which the rate
begins to decline due to the thermal denaturation of the
enzyme. This temperature is called the Optimum temperature
for that set of conditions. The results of such a study
for the transferase and hydrolytic activities are presented
in Figure 6. Phosphotransferase activity showed a greater
sensitivity to increased temperature, becoming increasingly
less active between 39°-62°C. In contrast, phosphomono-
esterase activity continually increased up to 62°C, the
highest temperature used. To test for enzyme instability,
"Purified Phosphotransferase" was exposed to temperatures
of either 50°C or 60°C in 0.1 M acetate buffer, pH 5.0.

At the indicated time (Table 2), an aliquot of the prepara-
tion was withdrawn and assayed for either phosphomono-
esterase or phosphotransferase activities by the standard
assay procedures. About 26% of the phosphomonoesterase
activity was lost during the first 15 minutes of exposure
at 50°C. This level of activity was retained over the next
15 minutes of exposure. Under the same conditions,
phosphotransferase activity was enhanced by 14% during the
first 15 minutes; afterwards 32% of the initial activity

was lost. At 60°C, complete loss of phosphotransferase

29

 

 

 

 

220 +
200
H 180 "24 a
g 1 -~20 a.
g 140 5
4,16
.3 120 .3
o 100 .
a -r12 2
so
:1- :1-
5 60 . 8 E
40
20 ‘ 4
1o 20 30 4o 50 60 70

Temperature (°C)

JFigure 6. Effect of increasing temperature on enzyme
(activities. Except for the reaction being run at the
.indicated temperatures, conditions for the determination
of transferase (Cl) and phosphatase '(0 ) were the same
as for the standard procedure ..

30

.HQ Hem
ceospoum mzo meHQEJE Hm u emeuemmcenuosmmosm How muu>uuom eucHOmodo

.Hc Hem
oemmeaeu mzm meHoE: v.o u emmueumeocoEocmmosm How muu>wuom eucHOmnme

 

0 mm 3

0 mm 3.

o 3 3

o S 3

o S . m .8

as E 3

a: E 3 .3
0.03 «.03 0 83:8

 

emeuemmceuuocmmocm emeueumeocosocmmocm Ameuccafiv

 

ensumnemsea
muu>uuo¢ e>uueaem eeua eusmomxm

 

.mucefiuuemxe floccuuuoce 03u CH cecueuoo euez mefluu>uuom cuoo you mecae> ueHuEum
.mufl>uuoe emmuosmmonm no emmuemmcenu Hecuue you Uohm um cemMmme one czencsuu3
cecu mmz uocvuae HE oa.o s .mHe>ueucH eEHu ceumowocu ecu you Doom no Doom necufle
ou cemomxe mes .o.m mm .ueumco eumueom 2 H.o cu =emeuewmcmuuocmmosm ceumuucm=

esa .muu>fluoe OHE>Nce co meucumuemaeu nous ou eucmomxe mo uoeMMMII.N mqmda

31

activity was noted after the first six minutes of
exposure. After this same time, 63% of the hydrolytic
activity was detected. Even after 30 minutes of exposure,
35% of the hydrolytic activity was retained.

The inactivation of phosphotransferase activity
was studied further in order to determine more accurately
the time course of transferase inactivation at 60°C. It
was also desirable to know if cytidine, the nucleoside
acceptor, could protect the phosphotransferase activity
from the denaturation caused by high temperature. The
transferase activity showed a slight activation during the
first two minutes of exposure at 60°C (Table 3). This
activation was followed by a precipitous drOp in activity,
complete inactivation being obtained between four and five
minutes of incubation. When cytidine (l umole/0.l ml)
was added to the enzyme during the incubation period, no
activation of enzyme activity was detected. Also,
cytidine, far from protecting the transferase activity,
seemed to increase the rate of heat inactivation.

Collectively, these results indicated that the
phosphotransferase activity was significantly more heat
labile than the hydrolytic activity.

Effects of Monovalent
and Divalent CatiOns

 

Since enzymes may have a specific metal ion

requirement, it was thought that studies on the effect of

32

TABLE 3.--Effect of cytidine on the activity of phosphotrans-

ferase exposed at 60°C. Conditions were as given in Table 2

except that cytidine (1 umole/0.l m1), when used, was added

to the enzyme preparation before exposing it to the elevated
temperature.

 

. Relative Activitya
Exposure Time

 

 

(minutes) Control + Cytidine
0 100 100
1 116 88
2 121 80
3 40 17
4 8 l7
5 0 7
6 0 0

 

aThe relative activity is based on the absolute
value for CMP production per hour at zero exposure time
(relative activity = 100). For this experiment this value
was 57 mumoles CMP produced per hour for the control and
54 mumoles CMP produced per hour when the preparation was
exposed at 60°C in the presence of cytidine.

33

various monovalent and divalent cations might give some
indication as to whether or not the same site was involved
in the transferase and phosphatase activities. Table 4
summarizes the results of experiments in which different
salts were added to the standard assays for phospho-
transferase and phosphomonoesterase activities. All salts

were added in the chloride form to give a final concentra-

tion of 3.3 X 10-4 M.

Monovalent ions increased both phosphotransferase
and phosphomonoesterase activities. No differences in the
effect of these ions were noted for the transferase activity.
All were equally capable of stimulating transferase activity,
giving a 23% increase in rate. However, the hydrolytic

activity was increasingly stimulated by the different ions

in the order Na+ > K+ > NH4+.

Both hydrolytic and transferase activities were

stimulated most by Mgz+. Both activities were equally

enhanced by Ca2+ and C02+. Mn2+ enhanced phosphomono-
esterase activity, but it inhibited the transferase
.reaction by 43%. Zn2+ also inhibited the transferase

activity; it had little or no effect on phosphomonoesterase.

Inhibition by EDTA and NaF

Since the experiments summarized in Table 4
indicated a possible requirement for a metal ion by both

transferase and hydrolytic activities, it was considered

34

TABLE 4.--Effect of various cations on enzymic activity.
All salts were prepared in the chloride form and the final
concentration used in the reaction mixture was 3.3 X 10'4M.

 

Relative Activitya

 

Cation Added Phosphomonoesterase Phosphotransferase

 

None 100 100

 

Monovalent Cations

 

 

NH4+2 136 ° 123
K+ 143 123
Na+ 175 123
Divalent Cations
Ca2+ 153 152
c62+ 156 152
Mgz+ 219 247
an+ 169 57
an+ 105 29

 

aRelative activity is expressed in relation to the
pNP produced per hour (0.37 umoles) or to the amount of
CMP produced per hour (15 mumoles) in the absence of added
salts (relative activity = 100).

 

 

 

 

that

wonl

cent
phos
Pho:

the

inj
occ
tra
con.
of 1

inhi

35

that substances which interfere with metal-enzyme complexes
would inhibit both enzymatic reactions.

Figure 7 illustrates the effect of variable con-
centrations of EDTA, a metal chelating agent, on both
phosphotransferase and phosphomonoesterase activities.
Phosphotransferase activity was inhibited very rapidly as
the concentration of EDTA was increased. NO activity was
present at a concentration of 6.6 X 10-5 M EDTA. At this
same concentration, 68% of the original phosphomonoesterase
was retained. Even at the highest concentration of EDTA

tested (3.3 x 10‘?

M), 7% of the phOSphomonoesterase
activity was retained.

Phosphomonoesterase activity was slightly
inhibited by NaF (Figure 8). Maximum inhibition (37%)
occurred at a concentration of 2 X 10-4 M. Phospho-
transferase was slightly enhanced by about 20% up to a F-
concentration of 1.53 X 10.4 M. Higher concentrations
of F- resulted in transferase inhibition. Maximum
inhibition of 20% was obtained at a F- concentration of
3.3 X 10‘4 M. This then, is the first instance in these
studies in which phosphomonoesterase activity was more

sensitive to inhibition than the transferase activity.

Effect of Denaturing Agents
The rationale for the experiment in this section
is as follows. Different enzymes at the same temperature,

pH, and ionic strength may be expected to behave differently

36

R/ R0
.9
_.|

0.2
.. T

O 1 4 ; n [HM—-

0.0 0.4 0.8 1.2 126 2.0 2.4 2.8 3.2
2)

 

 

Concentration of EDTA (M x 10"

Figure 7. Effect of‘ EDTA concentration on enzyme activity.
Assays for transferase (C1) and phosphatase (0) were
Conducted as described in the standard procedure except that
an appropriate aliquot of a 10‘3 M EDTA solution prepared in
0 - 10 M aCetate, pH 5.0, was addedto thereaction mixture
t<3 give the final concentrations of EDTA indicated in the
figure. The points used to prepare the curves represent
the averages obtained from three separate experiments.

The vertical lines indicate the 1: standard deviation

o1:>'I::.ained for each sample.‘

37

 

 

J l 4 1 n L A 1 l
7

0:4 0.8 1.2 1.6 220 224 2T8 3f2‘ 3Z6

Concentration of NaF (M x 10—2)

Flgure 8. Effect of sodium fluoride on enzyme activity.

e experiment was conducted as described in. Figure 7 except
that a 10'3 M NaF solution prepared in. 0.10 M acetate was
“sad. Phosphotransferase (D); phosphatase (0). Each point
represents the average frOm three, separate experiments.
Standard deViations are indicated by the vertical lines.

38

when subjected to an increasing concentration of a
denaturing agent since they are likely to be denatured at

different rates.

Effect of HqC12.--The behavior of the transferase

 

and hydrolytic activities was determined in the presence

of increasing concentrations of HgCl2 (Figure 9). It

should be noted that for this experiment hydrolytic

activity was determined by the release of Pi since the
addition of alkali for the determination of pNP caused the
formation of a yellow precipitate. Although the parallelism
in the loss of activities might suggest that an enzyme
possessing both transferase and hydrolytic activities was
being inhibited, an equally valid possibility is that the
inhibition was generalized denaturation and therefore the

results are equivocal.

Inactivation by Urea.--The two activities also

 

showed only slight differences in sensitivity to urea
(Figure 10). A slight activation for both hydrolytic and
transferase activities was observed at low urea concentra-
tion. As the concentration was increased, a gradual loss
of activity in both reactions occurred at approximately
the same rate. This suggested that both activities are

about equally sensitive to urea.

Effect of Organic Solvents.--Phosphotransferase

 

activity'was very sensitive to the presence of organic

solvents (Figure 11) . Alcohols were potent inhibitors,

R/Ro

39

 

 

 

 

 

1.01
0.81-
0.6
0.4.
0.2.,
~43\\\\\\fl3
: e 1 : 4 :‘_{}:_ ETC 5

0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6

Concentration of HgC12 (M x 104)

Figure 9. Effect of HgCl concentration on phospho-
transferase and phosphomonoesterase activities.
Transferase activity (E!) was determined by the
standard assay. Hydrolytic activity was assayed by
determining the amount of Pi released from pNPP (CD).
Concentrations of HgCl indicated in the figure were
obtained by the additign of an appropriate aliquot
of a solution of 10'3M HgCl prepared in 0.10 M
acetate, pH 5.0, to the rea tion mixture. Similar
results were obtained in two additional experiments.

R/Ro

40

 

0.2 I

 

: e
0 1 2
Molar Concentration of Urea

Figure 10. Activation and inactivation of enzyme
activity by urea. The molar concentration of urea
indicated in the figure was obtained by the addition
of an apprOpriate aliquot of an 8 M urea solution pre-
‘pared in 0.10 M acetate, pH 5.0, to the reaction
Inixture. The extent of the transferase (C3) and
hydrolytic activities (0) was determined by the
standard procedures. Similar results were obtained in
'two additional experiments.

R/Ro

R/Ro

41

2 5 A Butyl Alcohol B Methyl Alcohol

2.04,

1.5 .

1.0. 1

0.5V

2.5 _ C Acetone A D Ethylene Glycol

 

 

 

 

 

5 15 25 35

% Organic Solvent in Reaction Mixture

Figure 11. Effect of alcohols and acetone on enzyme
activity. The organic solvent was added to the enzyme
and both were kept on ice for 10 minutes before the.
addition of substrate to start the reaction. Similar
results were obtained in two additional experiments.

42

for example, butyl alcohol at 3.3% caused 100% inactivation
of the transferase reaction (Figure 11A). Phosphomonoester-
ase activity behaved markedly different. This activity

was significantly stimulated by the organic solvents
tested. In a concentration of 6.7% butyl alcohol, for
example, hydrolytic activity was increased by 96%. On

the other hand, transferase activity was activated by
ethylene glycol much more than phosphatase at a low con-
centration of the glycol (Figure 11D).

Kinetic Studies of Phosphotransferase
and’Phosphomonoesterase Activities

If the two activities are present at a single
site on the enzyme, then the Michaelis constant (Km) for
the phosphate donor in the transferase reaction and the
hydrolytic reaction would be expected to be the same. If
different enzymes or different sites are involved, then
the Km values for each activity might be different. Thus,
investigations were conducted to determine the Michaelis
constants for the two activities.

A Michaelis-Menten plot (30) for phosphotrans-
ferase activity with cytidine as the acceptor yielded the
(mxrve shown in Figure 12. The sigmoidal shape of the
cnxrve is characteristic of so-called "allosteric" enzymes
which possess multiple cooperative binding sites (31) .
Kinetics of this nature could be explained on the basis of

time subunit model described by Monod et a1. (32). Because

 

43

mu moles CMP/hr

100- /

 

l 2 3 4 5 6 7 8
[pNPP] M x 103

Figure 12. Effect of increasing amounts of phosphate
donor substrate on the velocity of phosphotransferase.
Variable amounts of pNPP were used as the_donor
substrate in this study, otherwise all other conditions
were as described in the standard procedure for the.
transferase assay.

44

of the nature of the concepts inherent in the model pro-

posed by Monod et al. and since the findings of many of the

experiments to be presented presently could be based on

the model, it seems appropriate to state the basic tenents:

(l)

(2)

(3)

(4)

(5)

(6)

Allosteric proteins are oligomers the protomers
of which are associated in such a way that they
all occupy equivalent positions. This implies
that the molecule possesses at least one axis
of symmetry.

To each ligand able to form a stereospecific
complex with the protein there corresponds one,
and only one, site on each protomer. In other
words, the symmetry of each set of stereo-
specific receptors is the same as the symmetry
of the molecule.

The conformation of each protomer is constrained
by its association with the other protomers.

Two (at least two) states are reversibly
accessible to allosteric oligomers. These states
differ by the distribution and/or energy of
inter-protomer bonds, and therefore also by the
conformational constraints imposed upon the
protomers.

As a result, the affinity of one (or several)

of the stereospecific sites toward the correspond-
ing ligand is altered when a transition occurs
from one to the other state.

When the protein goes from one state to another
state, its molecular symmetry (including the
symmetry of the conformational constraints
imposed upon each protomer) is conserved.

A great deal of the rest of this thesis then,

especially this section on kinetics, will be concerned with

the possibility that phosphotransferase and phosphomono-

esterase are polymeric. It is evident that if this is

true then the task of purifying and characterizing the

enzymes is increased .

45

The polymeric nature of enzymes has been shown,
in general, by demonstrating that they can be made to
associate or dissociate into subunits (the lowest level of
organization) or monomers (the level of organization
below oligomers).

The interaction of substrates with enzymes which
possess multiple cooperative binding sites is usually
assessed by noting the kinetic behavior of the enzyme in
the presence of substances known to cause association
or dissociation of proteins. Thus it was decided to look
at the transferase kinetics in the presence of salts
which have opposing effects on the polymeric structure of
proteins. Robinson and Jencks (33) studied the effects
of various salts on the solubility of acetyltetraglycine
ethyl ester (ATGEE), a model for peptide and amide groups
in protein. The results of this work, and that of Nagy
and Jencks (34) showed that the salts tested could be
placed in a series depending on their effects by three
criteria: (a) the effect on the solubility of ATGEE;

(b) inhibition of protein denaturation; and (c) promotion
of polymerization of F-actin. It was found, for example,
that CH3COO-, the anion used to prepare the buffer in the
present studies, effected an increase in the activity
coefficient of ATGEE (salt out) and favored the poly-
merization of F-actin. However, ClO4- caused an increase

in the solubility of ATGEE (salt in) and depolymerized

46

F-actin. CH3COO- thus acts as an associating anion, i.e.,
favors polymerization of protomers and 0104- acts as a
dissociating anion which favors the dissociation of an
oligomer into its constituent protomers. Accordingly,

the transferase kinetics as a function of increasing
donor concentration were studied with CH3COO- treated

and with C104- treated samples.

Since, with unlabelled cytidine as the acceptor,
it could be argued that the lag detected at low donor sub-
strate concentration was caused by the formation of
quantities of nucleotide too small to detect, 3H-uridine
was used in these studies to improve the sensitivity of
detecting small quantities of product.

As shown in Figure 13, the sigmoidal shape of the
v vs. [S] plots was realized both in CH3COO- and C104-
treated samples. However, in C104- treated samples, the
sigmoidicity of the curve was more pronounced. This fact
was confirmed by double reciprocal plots (35) wherein
the curved line determined from the data of samples
treated with C104- was significantly more concave upward
than the curve obtained for the CH3COO- treated samples
(Figure 14).

In instances when non-Michaelian kinetics such as
that given in Figure 14 is obtained, it has become common
practice to subject the data to Hill plots (36) in attempts

to describe the system more fully. Wyman (37) has referred

to the slope of this plot (n) as an interaction

CPM 3HUMP x 102/hr

47

 

 

50 .
O
40 "' /.
C
30 x
I
C
20
I
10 ,
I
I O
O
i z 5 I g 6 i ‘5

[pNPP] M x 103

Figure 13. Velocity of phosphotransferase as‘a function
of phosphate donor concentration obtained for acetate

and perchlorate treated samples. Separate enzyme prepara—
tions were dialyzed overnight (4°C) against either 0.1'M
acetate buffer (pH 5.0) or against the same acetate buffer
containing 0.05 M NaC104. —The reaction mixture consisted
of: 19 pg Protein; 20 u moles acetatg buffer, pH 5.0;
variable concentrations of pNPP and H-uridine (80,000
cpm) as the acceptor. When NaClO was present in the
reaction mixture it was used at a concentration of .05 M.
The velocity is expressed as cmp incorporated into H-UMP;
determined as deScribed in Materials and Methods.

CH3C00' treated (0); C104" treated (O).

x 103

C PM

300 "

 

48

l l l l A I
v v

T v t

2 4 6 8 10 12 l4 16 18 20

l

[pNPP]- (M’1 x 10‘

Figure 14. Double reciprocal plot for pNPP in the phos-
photransferase reaction. This plot was prepared from the
data given in Figure 13. CH3C00 treated ([3); treated,

with 0

.05 M C10 ' (O).

4

49

coefficient. An increase in the value of n away from unity
is taken as an indication of an increased interaction
between substrate-binding sites whereas a decrease in n is
taken as a weakening of the interaction between the

binding sites. Hill plots prepared from the data in

Figure 14 showed a change in lepe as the concentration

of donor was increased (Figure 15). This change occurred at
a concentration of pNPP of approximately 1.93 X 10"4 M

in both the CH3COO- and C104_ treated samples. However,
the Hill coefficients (n) were different. In the CH3COO-
treated samples the Hill coefficient increased from 1.10

to 1.55 while, in the C104- treated samples, n increased
from 1.2 to a value of 2.30.

The nonlinearity of the reciprocal plots and Hill
coefficients of greater than one suggested the possibility
for increased interaction of multiple donor sites (perhaps
2) in the transferase reaction. This interaction was
substantially strengthened by treatment with C104-.

When the concentration range of variable donor
phosphate utilized was higher than that noted above, the
sigmoidicity of the Michaelis—Menten plot was not
observed either in the C104- treated or CH3COO- treated
samples. A double reciprocal plot under these conditions
was best approximated by a straight line. Figure 16 shows
a typical plot produced with high concentrations of pNPP

in the absence of C104 from which an apparent Km of 3.1

t 0.3 mM was obtained.

50

0.8..

1

004‘
0.2“

log Vm-V

-O.&

I

.100“‘

 

 

-5 -4 -3
log [pNPP]

Figure 15. Hill plots of the phosphotransferase reaction.
These plots were prepared from the data given in Figure 13.
Data is plotted as log [v/Vm-V] as a function of log [pNPP] .
Hill coefficients obtained for the CH COO (E1) and

C104- (€>) treated samples are indicated in the figure.

51

.
_

Y»"‘.« .01 sun—nun.- my

cpM'l x 103

 

 

A
T—

1 2 3 4 5 6 7 8 9 10

_1
q-
-

1 -2

1(M- x10)

[pNPP]-

Figure 16. Reciprocal plots of phosphotransferase at high
pNPP concentrations. Conditions were as described in
IPigure 13 except that higher variable concentrations of
pNPP were used.

52

Michaelis-Menten plots (Figure 17) for hydrolytic
activity did not show a sigmoidal relationship over the
same range of phosphate ester concentrations used in the
transferase studies. A Michaelis constant of 2.4 i 0.5 mM
was determined from double reciprocal plots of the data
(Figure 18).

When uridine (1-2 umoles) was present during the
determinations, the initial velocity was decreased
(Figure 17). Double reciprocal plots indicated that the
uridine probably acted as a non-competitive inhibitor of
the hydrolytic reaction since the Km was not affected but
the V max was decreased (Figure 18).

Tha apparent difference in the Michaelis constants
obtained for transferase and hydrolytic activities sug-
gested that a simple one donor site—one acceptor site
model for both reactions could not be invoked. It is
possible that different enzymes or perhaps separate donor
sites in the same enzyme are responsible for each
activity.

Sedimentation in Sucrose
Density Gradients

An enzyme will sediment in a sucrose gradient
subjected to a centrifugal field at a rate which is
primarily dependent on its molecular Size. It is possible,
then, to separate a mixture of enzymes of different

molecular sizes by this technique.

V (mu moles pNP/hr)

53

500 L
T
4oo«L

300.

200" A

A
100T %”’r

 

l
r

l 2

[pNPP] (M x 103)

Figure 1?. Kinetic pattern of pNPP saturation in the
phosphomonoesterase reaction. Conditions were the

same as indicated in Figure 13. The velocity was
determined in the absence ((3) and presence of non-
labelled uridine. l u mole uridine in reaction mixture
(0); 2 p moles uridine in reaction mixture (A) .

54

201
18

16..
14 4» .

12-.

100

l/V x 103

4 4» /.
A

 

91 1 2 3
1 1 -3

[pNPP]- (M' x 10‘ )

Figure 18. Reciprocal plot for pNPP in the phosphOb
monoesteraSe reaction. The plot was prepared from the
data obtained in Figure 17. No added nucleoside (o );
1 14 mole uridine added (Q); 2 11 moles uridine

added (A) .

55

Consequently, if two different activities are
associated with the same enzyme, they should sediment to
the same location in the sucrose gradient upon centrifuga-
tion. If they are associated with differnt enzymes, they
probably would not sediment together since their location
in the gradient would depend on the molecular size of the
different enzymes responsible for each activity.

Figure 19 gives examples of sedimentation profiles
obtained for "Purified Phosphotransferase" preparations
centrifuged in sucrose density gradients buffered at
various pH values in 0.1 M acetate. In no case could
absolute and unequivocal separation of transferase and
hydrolytic activities be achieved. At pH 5.0 both
activities migrated as broad bands that were not entirely
superimposable. Upon lowering the pH to 4.5, each
activity was resolved into two or more bands and the
apparent rates of sedimentation were slower. At the
still lower pH of 3.0 transferase activity was resolved
into two nearly symmetrical peaks. The sedimentation
rate of the slower sedimenting band was even less than
that noted at pH 4.5. Likewise, the phosphomonoesterases
sedimented at slower rates; several bands which overlapped
but were not congruent with the transferase bands were
present.

Perhaps the most significant finding here was the

demonstration of the appearance of two distinct bands of

mu moles pNP/hr

56

 

 

 

 

 

  

 

, 2:7 A |
220 4. ‘ pH 5.0 i
O
x +5033
180. . I \\\.180
. I 8%
1’ l ' o T'40E§Ch
140.. I I mm140
2;”.
I
100" . 1:100'
' s s
4- __20
l I
60.. | 60.
.. . 4.10
20.. I - ' ' 2o ..
e g . 1 £>lgg
1 2
Volume in ml Volume in ml
100.. l
C pH 3.0 100
“ 80..
< 8° .2
a a
m 60.. 60 5
.3? a:
g 40.. 40 '3
1 E
a n
201. . 20 E

 

 

Volume in m1

Figure 19. Ultracentrifugation pattern of enzyme activities as
a function of pH.- "Purified Preparation" was exposed to 0.10 M
acetate buffered at either pH 3.0, 4.5, or 5.0 for three hours

at 4°C. It was then layered (121 ug protein) onto sucrose
gradients (5-20%) prepared with the acetate buffer at the same
pH to which it had been previously exposed. The gradientS‘were cen-
trifuged at 38,000 rpm for 31 hours at 2°C. The fractions
collected were diluted with 1 ml of 0.10 M acetate buffer, pH 5.0
and assayed for transferase (E3) or phosphatase activity (CD).
The figure shows the sedimentation profiles obtained for both
activities when contrifugation was carried out at pH 5.0 (A),

pH 4.5 (B) and pH 3.0 (C).

mu moles CMP/hr

57

slower sedimenting transferase activity in response to a
lowering of the pH. From an examination of the profiles
it can be said that it is probable that some of the
phosphatases do not have transferase activity. It is
equally possible for the transferases to have hydrolytic
activity associated with them.

The shift to slower sedimenting forms in response
to a lowering of the pH suggested that an oligomeric
macromolecule, possibly possessing both transferase and
hydrolytic activities, was dissociated into its con-
stituent subunits. Various oligomeric enzymes, e.g.,
aldolase (38), are dissociated as the net charge on the
oligomer is altered by a variation of the pH. Several
denaturants have also been used to cause the dissociation
of oligomeric enzymes (39). For example, Cottam eE_al.
(40) made use of urea to dissociate pyruvate kinase
(M.W. 237,000) into 4 subunits of molecular weight
57,200.

Figure 20 shows the sedimentation profile obtained
when "Purified Phosphotransferase" was centrifuged in the
1 presence of 3 M urea in a sucrose gradient buffered at
pH 5.0. This profile resembled somewhat the sedimentation
profile obtained in Figure 19C wherein centrifugation was
carried out at low pH, indicating that urea can also cause
the formation of slower sedimenting forms. It should be

recalled that earlier only one major band for both

58

 

 

 

 

 

 

73.0 67.2

60.1.. N" ’ 1L60

50 .. .50 H
s Q
E H
z 40 -- 4,40 U
Q In
3 3
H 30 J. ..30 0
O E
a :-
:1 E
E 20 ., 120

10 .- ”10

I I I I
1

 

 

 

Volume (ml)

Figure 20. Effect'of urea on the sedimentation profiles of
Phosphotransferase and phosphomonoesterase. The preparation
"as exposed to 3 M urea for 2 hours at room temperature and
then applied (121 pg protein) to a sucrose gradient (5-20%)
buffered with 0.1 M acetate, pH 5.0 containing 3 M urea.
C3gtitrrifugation was carried out at 38,000 rpm for 31 hours at
2 C. Fractions collected were diluted with lyml of acetate,
PH 5.0 and then analyzed for enzyme activity. Phosphotrans-
ferase ( D ) ; phosphomonoesterase ( o ) .

59

activities was obtained at pH 5.0 in the absence of urea
(Figure 19A).

The results suggested that both transferase and
phosphomonoesterase activities could be associated with
an oligomeric enzyme. This suggestion was derived from
the fact that both a lowering of the pH from 5.0 in
acetate buffer as well as the presence of 3 M urea at
pH 5.0 in the gradients caused the appearance of slower
sedimenting forms for both activities. It did not appear
that these new forms represented the dissociation of the
same oligomeric enzyme into lower orders of polymerization
since congruence between the peaks for each activity was
not observed. Additionally, the resolution of new
phosphatase peaks not possessing transferase activity
argued in favor of separate proteins for each reaction.
However, evidence will now be presented which suggested
that both activities may indeed be associated with the
same enzyme.

Evidence that Phosphotransferase
Possesses Hydrolytic Activity

 

As mentioned above, the occurrence of several
transferase and hydrolytic activities sedimenting at
different rates in the sucrose density gradients strongly
suggested that both activities were associated with a
dissociable oligomeric enzyme. The appearance of more

than the expected number of slowly sedimenting forms,

60

however, offered the possibility that something other

than dissociation was taking place. Perhaps random
breakage of the molecule due to the low concentration of
protein applied could account for some of the newly formed
species, especially in the case of the hydrolytic activity.
To circumvent this possibility, Sephadex G-200 columns
were employed so that more protein could be used without
suffering a great sacrifice in resolution.

An example of the elution profile obtained from
three separate experiments is shown in Figure 21. Three
major bands of transferase activity (Bands I, II and III)
were eluted from the column accompanied by superimposable
bands of phOSphomonoesterase activity. Again, as in the
sucrose gradients, other hydrolytic bands were resolved
which were not associated with any transferase bands.

An elution profile obtained for hydrolytic activity with
CMP as substrate exactly paralleled the phosphomonoesterase
profile obtained with pNPP. Thus, the enzyme responsible
for the hydrolytic activity could act as a nucleotidase.

The approximate molecular weight of bands I, II
and III was determined from a plot of elution volume vs.
the log of the molecular weight of the reference marker
as indicated in Figure 22. The molecular weight obtained
for each band is given in Table 5.

The values obtained for the molecular weights

raised the possibility that the transferases and

61

 

 

 

 

 

 

III
.51 mp moles
240- ' .40
I II
’ o
0
H180" -30 .u
€ 0 ' fi
n. I n.
z 5
n‘ o
a: .- - a:
3 H
s - 2
2120.» . ..20 :1
I [J ’ E
I I
I C
I
60- . ’ +10
I
o
. O
I
O
a a 5-: . . .. ’
10 20 30

Fraction Number

Figure 21. Chromatography of "Purified Phosphotransferase"
on a Sephadex G-200 column. "Purified Preparation" (14 mg
protein in 2 ml 0.10 M acetate, pH 5.2) was applied to a
Sephadex G-200 column (45 x 2 cm) and eluted with 0.1 M
acetate, pH 5.2. A flow rate of approximately 10 ml per
hour was-utilized and fractions of 4.0 ml were collected.
Aliquots were withdrawn and assayed for transferase (C3)

or hydrolytic activity (0).

62

O

\ Blue Dextran
\

\

+
/

   
 
 
     
   

Apoferritin

Catalase

Nunbmmflmmo
l I

.. Band I—e—o

Band II —-9
Alkaline Phosphatase
10 Jr-

92F
3..
7d:-

Molecular Weight

Hemoglobin

Band III —0

51)
41-
3...

2..
Cytochrome-C

 

lo : A n 1 : 1 1 I 1 n L J

10 20 30 40 50 60 70 80 90 100 110 120 130

Elution Volume (m1)

Figure 22.~ Determination of molecular weights of Phospho-
transferase by chromatography on Sephadex G-200. Standards
of kn0wn molecular weight were eluted from the Sephadex
cOlumn under the same conditions used for the "Purified
Preparation". The elution volume (Ve) of each standard was
determined as described in Materials and Methods.) The arrows
shown in the figure indicate the molecular weight and elution
volume determined for the transferase bands eluted from the
column.

63

TABLE 5.--Approximate molecular weight of three phospho-
transferase bands eluted from a Sephadex G-200 column.

 

 

Band Molecular Weight i Std. Dev.
I 2.0 x 105 i 0.45 x 105
II 1.0 x 105 r 0.23 x 105

III 5.3 x 104 t 1.20 x 104

 

corresponding phosphatases eluted from the column
represented tetrameric, dimeric, and monomeric forms of
the same enzyme. The basic subunit of the enzyme might

be represented by Band III which has an apparent molecular
weight of 5.3 X 104.

Sucrose density gradient analysis of the "Purified
Preparation" in which a larger amount of protein (3.0 mg)
was used as well as a slightly higher pH (5.2) produced
three or four forms of both phosphotransferase and at
least as many forms of phosphomonoesterase activities
(Figure 23). Hydrolytic bands and the apparent correspond-
ing transferase band are designated by the same Roman
numeral. Thus in Figure 23, four hydrolytic bands are
depicted which are possibly associated with transferase
activity. There was a definite correlation in the
sedimentation rate for both activities in the fastest
sedimenting bands thus suggesting that single proteins

are responsible for both activities. The slower

mp moles pNP/hr

 

 

 

64

 

 

II P
200 $ . ..50
180. I.P . ‘
160 .L . ' if 40
) I T IV P E
140 (+- . \
II P g;
1 Q U
120 a * ->30 m
w
'3
O o
100 .. E
2
II T 0 ‘
80 Y I 0 0 <~20
I
IV T
60 H) I
‘ I
III T
40 it I I .. $10
20 .
I I I '
I n q
. .g s ,L L 4.. .'."5‘
0.5 l 1.5 2.0
Volume in ml
Figure 23. Ultracentrifugation pattern of "Purified

Phosphotransferase" in sucrose gradients at pH 5.2 in
0.1 M acetate. "Purified Phosphotransferase" (3mg
protein) was layered on sucrose gradients (5-20%) and
centrifuged at 38,000 rpm for 34 hours at 2°C. Frac-
tions were collected and assayed as described in
Figure 19. Transferase ([3); phosphatase ((1).

 

 

 

pk
no
mi1
COL
rep
men
thi:
eac}

PhOs

65

sedimenting bands of transferase and phosphatase activity
showed a very poor correspondence of sedimentation rates.
Compare, for example, Bands II with Bands IV.

Although a direct comparison between this run and
those depicted in Figure 19 cannot be made since more
protein was used and the time of centrifugation was
slightly longer in the present study, nevertheless, it
is likely that the slowest sedimenting bands in Figure 23
(Bands IV) may be identical to the slower sedimenting
forms which appeared at low pH (Figure 19). Note that at
pH 3.0, the transferase band and hydrolytic band also did
not coincide. Apparently, there must be a certain
minimum size at which both activities remain associated.
Could it be possible that the 53,000 molecular weight form
represents this minimum? If so, then the slower sedi-
menting forms could result from the dissociation of
this protomer (monomer) into two non-identical subunits
each solely responsible for either hydrolytic or

phosphotransferase activity.

 

8550

DISCUSSION

It should be stated candidly at the outset of this

discussion that the original objective of this study was

 

not completely realized. This must be said in order to
provide some perspective to the original goals and in

order to assess the findings and their significances as

.—F'-_‘
_\: ~ ' .

well as to suggest possible studies in the future.

It has been demonstrated here that the nucleoside
phosphotransferase preparations obtained from carrot
leaves possess phosphomonoesterase activity in all stages
of purification. It has been suggested that both
activities may be associated with the same protein(s).

The behavior of the "Purified Phosphotransferase" prepara-
tion under a number of experimental conditions provided
support for this conclusion.

The first suggestion that both activities may be
associated with the same enzyme was obtained from attempts
to separate the transferase activity from the hydrolytic
activity on ion exchange columns. The fact that both
activities apparently retained a similar charge distribu-
tion over a wide range of elution conditions suggested the
possibility that both activities might be the result of
the same protein (Figures 1 and 2). Additional support

was obtained when it was discovered that the activities
66

67

could not be separated either by molecular seive
chromatography (Figure 21) or by centrifugation in
sucrose density gradients (Figure 23), two techniques
which discriminate proteins on the basis of differences
in size.

The concept of an enzyme possessing both phospho-
transferase and hydrolytic activities is not unique.
Georgatos (41), for example, has described a human

placental alkaline phosphatase that had been purified

 

450 fold which exhibited phosphotransferase activity. 1
However, it should be noted that sufficient data were not
given from which one could state unequivocally that the
transferase and phosphatase activities did indeed reside
on the same protein.

In the present studies gel filtration on Sephadex
G-200 columns (Figure 21) indicated the presence of
multiple molecular forms of enzymes responsible for
both activities. Molecular weight determinations (Table 5)
indicated that these forms probably represented oligomeric
and protomeric forms of the enzyme. It seems unlikely at
this stage of purification that multiple forms of proteins
possessing both transferase and hydrolytic activities could
exhibit maximum activity in the same fractions unless the
same enzymes are responsible for both reactions.

Cursory examination of the sucrose gradients with

hemoglobin as the reference standard indicated that the

68

molecular weight of the slowest sedimenting forms
apparently did not exceed 50,000 (data not reported).
Earlier in this thesis it was suggested that these smallest
sedimenting forms for both activities may represent the

dissociation of catalytically active, non-identical sub-

 

units which, together, comprise the native enzyme. One
subunit could be responsible for the hydrolytic function
of the enzyme while the second, smaller subunit would

contain the nucleoside phosphotransferase activity. The

 

differences in various biochemical parameters noted in
this study could then be explained on the basis of a dif-
ference in the stability and the properties of each of
these discrete subunits. Of course, it is just as likely
that these forms represented the dissociation of subunits
from totally different enzymes.

The kinetic data for the transferase reaction
showed a transition from Michaelis-Menten kinetics to
sigmoidal kinetics as a function of pNPP concentration.
Maximum transition was obtained with enzyme treated with
NaClO4 (0.05 M) wherein the Hill coefficient (n) increased
from 1.20 to 2.30 (Figure 15). According to the model
preposed by Monod gt_31. (32), such an increase of the
Hill coefficient argues in favor of an increased inter-
action between multiple substrate sites, possibly two.
This interaction could occur by association of subunits,

by a conformational change of the protein molecule or by

69

both. However, since the claim has been made here that
both activities might be associated with the same oligomeric
enzyme, one would expect to obtain sigmoidal kinetics for
the hydrolytic function of the enzyme as well. This was
not the case as all double reciprocal plots made for
hydrolytic activity were best approximated by straight
lines. The possibility exists, then, that the multiple
molecular forms eluted from the Sephadex G-200 column
(Figure 21) were produced by the aggregation of an active
form of the enzyme (M.W. 53,000) caused by the par-
ticular conditions employed in the investigation. This
does not detract from the fact that an enzyme possessing
both activities was involved in the aggregation into
polymeric forms. An equally valid possibility to explain
the kinetics obtained for the phosphotransferase activity
then is that it reflects a competition for the donor sub-
strate between a hydrolytic "site" and a transfer "site"
which is not expressed in the associated state.

A precedent for a suggestion of two sites binding
donor substrate for phosphotransferase enzyme exists.
During the writing of this thesis, Brunngraber and Chargaff
(42) published a paper in which evidence was given which
suggested the presence of two hydrolytic sites binding
pNPP’in the carrot root phosphotransferase. This sug-
(gestion was based on two major lines of evidence: (a)

rmntlinearity of double reciprocal plots in the hydrolysis

 

r

In.“

 

 

 

70

of pNPP and ribose-S-phosphate, and (b) the presence of
two ionizing groups in the free enzyme which could bind
pNPP. The suggested model was composed of two hydrolytic
sites and one acceptor site. "One hydrolytic (transfer)
site and the acceptor (water) site remain in the same
relation to each other as in the two-center model." It
was also found that uridine, the acceptor nucleoside used
in these studies, impeded the ". . . hydrolysis of the
second hydrolytic center which is incapable of transfer"
by acting as a possible non-competitive inhibitor of the
hydrolytic reaction. In the present study, uridine was
also found to non-competitively inhibit the phosphatase
activity of the leaf preparation albeit to a very limited
extent.

As mentioned above, in the present investigations
with the leaf preparation nonlinearity of double reciprocal
plots for hydrolytic activity was not observed (Figure 18)
suggesting that only one "site" (apparent Km = 2.4 I 0.5
mM) is responsible for the hydrolysis of pNPP. A second
"site" (apparent Km = 3.1 i 0.3 mM) could then function in
the nucleoside phosphotransferase reaction. It is not
positively known if this transferase site can also
catalyze the hydrolase reaction. The kinetic data and
sucrose density profiles obtained at low pH (Figure 19)
and in the presence of urea (Figure 20) would suggest that

it does not. Additionally, experiments in which organic

71

solvents were added to the reaction mixture (Figure llA-D)
also suggested that, even if both activities reside on the
same protein, there may be different transferring and
hydrolytic "sites." An alternative explanation would be
that the alcohols themselves are competing with the nucleo-
side in the case of the transferase reaction. This would
not, however, explain the action of acetone on the two
activities (Figure 11C).

In the present investigation, no attempt was made
to ascertain the number and kinds of acceptor sites. It
is suggested, however, that separate sites for water and
the nucleoside acceptor may exist if two non-identical
subunits described above do indeed comprise the native
enzyme. Thus, one donor site (hydrolytic) and a water
site would be found on one subunit. The second smaller
subunit would contain the second donor site (transferase)
and a nucleoside acceptor site. More evidence, especially
in regard to the acceptor sites, is needed to firmly
establish this hypothesis.

Besides the difference in double reciprocal plots
for the hydrolytic function, other differences were noted
between the leaf preparation and the root preparation of
Brunngraber and Chargaff. Thus, in addition to the dif-
ference in pH Optima previously described, no sigmoidicity
xmas observed in the kinetics of the transferase function.

It is possible that too high a concentration of pNPP was

 

 

 

 

72

used in their studies so that sigmoidal kinetics was now
obtained. It may be that one significant difference
between the two preparations is that the root enzyme does
not aggregate into large molecular weight forms, at least
Brunngraber found no evidence that it did. Could it be
possible that the carrot has "selectively" chosen to
produce an oligomeric phosphotransferase-phosphomonoesterase @-
enzyme in the leaf wherein many organic phosphate esters E

are produced in the process of photosynthesis? At present, ;

 

it cannot be said with any real degree of certainty that
the enzymes are not the same since somewhat different con-
ditions have been utilized in obtaining each preparation.
However, this is an intriguing possibility and further
investigations in an attempt to answer this question are
certainly warranted.

In summary, the most significant findings of this
part of the thesis are:

1. The nucleoside phosphotransferase and
phosphomonoesterase activities prepared from carrot leaves
could not be separated by physical and chemical procedures
utilized.

2. Multiple molecular forms of enzymes possessing
both activities were detected by elution from a Sephadex
G-200 column and by sedimentation in sucrose density

gradients.

73

3. Both activities could be dissociated into non-
identical slower sedimenting forms in sucrose density
gradients at pH < 5 and in the presence of urea (3 M).

The molecular weights of these slower sedimenting forms
were probably less than 50,000.

4. Transferase activity differed greatly from
phosphatase activity in sensitivity to heat, to denaturants,
to inhibitors and stimulators and to pH.

5. Kinetic data indicated the possible presence of
two sites binding pNPP in the transferase reaction; only
one site binding pNPP was detected in the phosphatase
reaction. In addition, uridine acted as a possible non-
competetive inhibitor of the hydrolytic reaction.

The possibility that an enzyme possessing both
activities on different dissociable subunits must be
considered.

In view of the findings described herein, it is
probable that the original objective of this section
of the thesis was set prematurely owing to the possibility
Of multiple forms of the enzyme(s). It is also possible
that even the purest preparation used was not as pure as
it was thought to be. Certainly, several additional
purification steps, such as the technique of isoelectric
focusing, could be included in future purification schemes.
Additionally, in further attempts to purify the phospho-

transferase from the leaf care should be taken to utilize

74

conditions under which the enzyme will remain primarily
in one form. A more complete study should also be made
to determine if, indeed, both activities are associated

with an identical oligomeric protein(s) .

SECTION II

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

75

INTRODUCTION

The role of 3',5'-cyclic adenosine monOphosphate

(CAMP) as an intracellular second messenger was first
established in liver glycogenolysis by Rall 3:31. (43).
Since then this compound has been shown to act as a
mediator for a number of animal hormones and to influence
a wide variety of cellular processes in many different
tissues and organs (44). The level of cAMP in the cell
is maintained by at least two enzymes. Adenyl cyclase
(45) catalyzes the formation of the cyclic nucleotide
from ATP. A phosphodiesterase hydrolyzes the cyclic
nucleotide to the monoester (46). Since this enzyme has
been found to be more widely distributed in cells than
adenyl cyclase, more work has been documented for the
PhOSphodiesterase.

Sutherland and Rall (46) provided the first
indication that cAMP could be inactivated. The enzyme
was demonstrated to be present in beef heart and the
Product was shown to be 5'—adenosine monophosphate
(5"AMP). Butcher and Sutherland (44) emphasized the
overall distribution of the cyclic nucleotide phophodi~
e$terase in various tissues of the dog where the action
Of CAMP has been documented. The properties of various

PhOSphodiesterases have been described; the enzyme from

76

77

beef heart by Butcher and Sutherland (47) , from dog heart
by Nair (48) , from rat brain by Cheung (49) and Cheung

and Sulganicoff (50) from rabbit brain by Drummond and
Perrot-Yee (51) and from rat liver by Menahan _e_1_:__a__l_. (52).
A phosphodiesterase has been described from at least one
unicellular organism; Chang (53) characterized a diesterase
produced by the slime mold Dictyostelium discoideum which
apparently acts as an acrasinase (54) since it destroys
the chemical substance (acrasin) which causes aggregation
of the motile, unicellular amoebae thereby producing small
multicellular fruiting bodies. This substance has been
shown by Konijn _e_t__a_l_. (55) and Konijn _e_t__a_l_. (56) to be
3' ,5'-cAMP. In general, the phosphodiesterases isolated
from most animal sources have a dependency on added Mgz+
for maximal activity, are stimulated by imidazole and are
inhibited by methyl xanthines. Additionally, Honda and
Imamura (57) have shown that partially purified phosphodi-
esterases from beef heart and rabbit brain cortex are
inhibited by phenothiazine and reserpine derivatives.
MiChaelis constants with cAMP as substrate vary from
approximately 0.10 to 0.60 mM. Recently, Cheung (58)
indicated the presence of an activator for the phosphodi-
eSterase from a crude homogenate of bovine brain cerebra.
The activator was shown to be a protein of molecular weight

0f about 40,000. The enzyme isolated from Dictyostelium

is apparently different from the animal enzymes since it

78

is not inhibited by caffeine. It also exhibits a somewhat

larger Km (2 mM) with cAMP as substrate.
There are no reports, to my knowledge, concerning

phOSphodiesterases from higher plants which hydrolyze

3',5' cyclic phosphate esters. As a result of the recent

demonstration of the effect of gibberellic acid on the

conversion of adenine-8-C into camp in barley aleurone

layers by Pollard (59), it seemed that such an investigation

was desirable. Certainly, if cAMP acts as a second mes-

senger in higher plants for one or more of the plant
hormones one would expect to find a mechanism for its
inactivation. Since the crude preparation prepared
during investigation of the nucleoside phosphotransferase
Vvas known to be very rich in phosphatase activity, it was
(decided to search for the enzyme in this preparation. It
61150 seemed desirable to determine the kinds of organic
E>hosphate esters that could be hydrolyzed by the crude

Eireparation. The investigations reported in this section

<>f the thesis, then, have a two-fold objective: (a) to

investigate the Specificity of the crude preparation
'tCNMard phosphate esters, since non-specific phosphatases
aJfie potentially useful in the identification of phosphate
esrters and (b) to make a cursory examination of the
IPIRDperties of 3',5' cyclic nucleotide phosphodiesterase,

arI enzyme that appears to occupy a central role in

79

Iregulating hormonal action in higher organisms and which

luas not been documented to occur in higher plants.

MATERIALS AND METHODS

Enzyme Source

 

The crude preparation described in Section I
was used in all studies. It was dialyzed for four hours
against two liters of cold distilled water (4°C) before

use.

Chemicals

 

Cyclic nucleotides were purchased from Sigma
Chennical Company, St. Louis, Missouri and were used
Witflnout further purification. Other chemicals and
reagents were of the highest purity available and were

also used without further purification.

Enzyme Assays

 

When substrates other than cyclic nucleotides
Werfle used the reaction mixture consisted of the following:
2~5mg of protein, 1 umole of organic phosphate ester,

0-(31. umole MgCl and 30 umoles sodium acetate buffer,

2
PH 55.2, in a final volume of 0.61 ml. The reaction
miIr‘tture was incubated at 45°C for thirty minutes and the
reeuztion stOpped by boiling it for five minutes. The

“darture was then cooled in ice and 0.10 ml of cold 5%

trichloroacetic acid was added to insure complete

80

81

precipitation of protein. The tubes were centrifuged for
five minutes with a clinical centrifuge set at 3/4
maximum speed. A 0.40 ml aliquot was withdrawn to which
0.20 ml of glass distilled water was added. The amount of
phosphate in this mixture was assayed according to the
method described by Ames (27) . Controls which were
identical to the experimentals were run except that sub-
strate was added immediately before boiling. All
spectrOphotometric readings were made at 730 run against
the control sample. Activity is expressed as mumoles
phOSphate released per hour.

One method that can be used to follow the action
of phosphodiesterase upon cyclic nucleotide monophosphate
(CNMP) was described by Butcher and Sutherland (47). The

basis of the method is:

CNMP Phosphodiesterasek NMP Nucleotidasem Pi + Nucleoside
' Tcommercial) '

The assay can be conducted with the source of
Phosphodiesterase and the added commerical nucleotidase
incubated together or it can be terminated at the NMP
level and then carried to completion by the addition of the
nucleotidase. Activity is measured by determining the Pi
released by the nucleotidase.

Preliminary studies showed that the crude prepara-
tion contained very high levels of both 3'- and 5'-

nucleotidase. Since the preparations released phosphate

82

from 3'- and 5'-AMP at 12-20 times the rate at which
phOSphate was released from cAMP, and since there was

no appreciable accumulation of the monOphosphates when

the cyclic ester was used as a substrate, it was obvious

that simply estimating phosphate release from the cyclic
ester would be an effective assay for phosphodiesterase

since this would be the rate-limiting reaction for

'phosphate release. Thus, the reaction mixture consisted

(Df the following: 10 mg protein, 1 umole cNMP, 0.1 U

Inole MgCl2 and 30 umoles of sodium acetate buffer of pH

5.2 in a final volume of 0.61 ml. The mixture was

:incubated at 45°C for two hours, at which time the

Jreaction was stOpped by boiling for 5 minutes. Phosphate

Ireleased was measured as described above. Controls were

rain and expression of activity is as described above for

non- cNMP substrates .

Since more than one enzyme is involved in the
(atssay for the degradation of cyclic nucleotides, studies
titzilizing cyclic nucleotide substrates will make reference
tn: 'bhe phosphodiesterase system rather than the phosphodi-

esterase enzyme since any treatments may affect one or

more of the enzymes involved in the assay.

Determination of Nucleotide Intermediate
The reaction mixture used to determine the identity

(If ruacleotide formed by the carrot diesterase consisted of

the following: 0.10 mg protein, 0.01 Umole MgClz, l u

83
mole cAMP, 2 umoles fructose 1:6 diphosphate and 20 u

moles sodium acetate buffer, pH 5.2, in a final volume of

0.41 ml. Individual tubes containing this mixture were

incubated for 10, 20 or 40 minutes and were then boiled

for 5 minutes to stOp the reaction. The tubes were then

centrifuged for 10 minutes at 3/4 maximum speed in a

clinical centrifuge. The supernatant solutions from all

the tubes were combined and the entire mixture streaked

con Whatman No. l chromatography paper which had been

Ipreviously impregnated with 10% (NH4)ZSO4. Commercial

:3amples of 5'-AMP, 3'-AMP, cAMP, adenine and adenosine
vvere also applied to a second sheet of chromatography

gpaper. The chromatograms were developed by ascending

tzechnique with 75% ethanol and the UV absorbing areas

lxocated with a UV light source. The UV absorbing areas

<2cxrresponding to the standards were eluted from the paper

and rechromatographed by thin layer technique on Silica

ggeell GF plates (2.5 cm X 7.5 cm). The chromatograms were

developed in a solvent system containing iSOprOpanol-

nuetflaanol-NH4OH and water (10:1:5:2 by volume) along with

cxbnunercial standards. The compounds were located as

given above .

Sucrose Density Gradients

Density gradients were prepared as described

earlier. Protein (15 mg) was layered on the gradients

and Centrifugation was carried out at 30,500 rpm for 22

84

hours at 4°C. Fractions were collected as previously
described and were diluted by the addition of 0.40 ml of
0.10 M acetate buffer pH 5.0. An aliquot of 0.01 ml of
each fraction was incubated with an equal volume of

acetate buffer containing either 3',5‘-cAMP or 2',3'-

cAMP (both 1.5 mg/ml) for 10 hours at 37°C. The reaction
Inas stOpped by the addition of 1.7 ml of the assay reagent
tised for the determination of phosphate. The sedimentation

xprofile is plotted as OD determined at 730 nm vs. the

:fraction number.

RESULTS

Substrate Specificity of Crude
Phosphotransferase

The crude preparation. 'was shown to contain at
lxeast several distinguishable enzymes that could hydrolyze
pflNPP (Figure 1). Consequently, it was decided to determine
time kinds of phosphate esters that could be hydrolyzed by
'tflis preparation. Since very few higher plant phosphatases
xvigth high substrate specificity have been described, an
easily obtainable crude preparation of known substrate
Specificity may be valuable as a source of such enzymes.
Afihflitionally such a preparation could prove of inestimable
Value in the identification and proof of structure of
newly-isolated phosphate compounds.

The results summarized in Table 6 indicate that
the crude preparation was capable of hydrolyzing a large
nuunber of phosphate esters (all substrates tested were
hYdrolyzed with the exception of Vitamin B12) , some con-
'taianing pyrophosphate bonds, to inorganic phOSphate. Of
‘najCIr significance was the observation that the phosphodi-
estner linkage of Bis-pNPP could also be hydrolyzed to
FUNDSphate. Since no literature on the hydrolysis of the
Phosphodiester linkage of 3' ,5'-cyc1ic nucleotides by plant

Preparations could be found, it was decided to determine if

85

86

TABLE 6.—-Relative apparent rates of hydrolysis of various
organic phosphate esters by "Crude Phosphotransferase" from
carrot leaves. Individual controls were run with each

 

 

phosphate ester listed in the table. The values given are
the averages for two separate experiments.

Phosphate Ester Mumoles Pi/hr.
Coenzyme A 2858
Deoxy-5'-Adenosine Diphosphate 2010
Pyridoxal Phosphate 1875
3—Phosphoglycerate 1670
Adenosine-S ' -Tetraphosphate 1608
Flavin Mononucleotide 1608
Fructose-1,6-Diphosphate 1320
NADP (TPN) 1252
Phosphoenolpyruvate 1250
Deoxy-5'-Guanidine Monophosphate 1195
Dihydroxyacetone Phosphate 1070
o—Phosphoethanolamine 1025
Thiamine Pyrophosphate 612
3 ' -Adenosine Monophosphate 585
Fructose-6-Phosphate 558
NAD (DPN) 535
Cytidine-2 ' , 5 ' -Diphosphate 508
2 ' -Adenosine MonOphosphate 492
Fruc tose-l-Phosphate 458
Flavin Adenine Dinucleotide 415
Rlbose-S-Phosphate 392
6‘fPhosphogluconate 362
Bl£3---p--Nitrophenylphosphate 342
GlLIcose—6-Phosphate 342
IE’henophthalein Diphosphate 302
5 ' ~Adenosine Monophosphate 258
Glyceraldehyde-3—Phosphate 128
G}Ucose-l-Phosphate 100
Vltamin B 0

12

 

 

87

'this preparation could act on this kind of substrate also.
'Table 7 lists, in decreasing order, the relative activity
cobtained with different cyclic phosphate esters. All the
<3yclic nucleotide substrates tried were successfully
hydrolyzed by the preparation thus indicating that a system
for the inactivation of cAMP and other 3',5'-cyclic
nucleotides indeed exists in higher plants. The results
from an investigation of the properties of this system
from the carrot leaf preparation will now be given.

Some Properties of the Cyclic

Nucleotide Phosphodiesterase
System from Carrot Leaves

lime Course

The release of phosphate from c dibutyryl AMP was
ffiound to be essentially linear for the first hour of
iancubation at 45°C (Figure 24). However, in order to
Cibtain significant activity, a two hour incubation period
Vvas generally used in these studies. During the second
kuour the rate of phosphate release falls off only very

S lightly .

Efoect of Temperature

Figure 25 shows the activity of the preparation
tuawards cAMP as a function of increasing temperature. No
apparent optimum temperature was obtained under the assay
C30nditions used, even up to 55°C. It was concluded that

time diesterase system could be studied at elevated

88

 

n.am Amzuoc mumcmmocmocofi wcwcaumo owaomo|.m..m

 

m.mm Amadoc mumcmmosmocoa mcamococm oaaomou.m..m
o.ma Amza Hmumusnflc 0c opwcmmocmocoe mnemococm Hmuxusnac oaaowon.m..m
N.ov Amzaoc mpmcmmocmocofi mcacaemcu owaoaou.m..m
0.0m AmzHov ouwcmmocmocofi mnemocfi oHHomou.m..m
uc\am mmH081z wcauomaosz oaaomu

 

.mponumz com

wamflumumz ca confluomwc mamsofl>mum muscoooum UHM©GMpm 0:» cu mcwcnooom coma mumB
mcoaumcHEnoumo .mm>mmH uouumo Eoum emmmammmcmuuocmmocm mcunu: an mwumuvaSm
ocfluomaosc oaaomo msoaum> mo mawwaoncmc mo mmumu uconmmmm 0>Humammll.h mqm<9

mu moles Pi/hr

89

 

 

O

50 --

40 +

O

30 -. O

20 -.

10 T

30 60 90 120 150

Time (minutes)

Figure 24. Time course of the formation of Pi by the crude
phosphodiesterase preparation. Reaction mixtures were pre-
pared as described for the standard procedure. At the
indicated time intervals, tubes were withdrawn and the con-
tents assayed for Pi as in the standard procedure. The .
8:5‘3l1\11;18:.il.‘.rate employed for this investigation was dibutyrl

c O

 

90

4O ..

w
O

my moles Pi/hr

ZO-A

10...

 

 

5 15 25 35 45 55

Temperature (°C)

Figure 25. Phosphodiesterase activity as a function of
increasing temperature. Enzyme and substrate (cAMP) were first
exposed to the indicated temperatures for 5 minutes to insure
that the. reaction was carried out at the desired temperature
for the entire period of incubation. Substrate .was added to

the enzyme to initiate thereaction after this pre-exposure
Period.

91

temperature without fear of 1033 of activity. Thus, in
most studies, an incubation temperature of 45°C was used
so that a significant amount of phosphate could be

released without resorting to prolonged periods of incu-

bation.

pH Optimum

An examination of the hydrolysis of cAMP in
acetate buffer of various pH values produced the results
given in Figure 26. Unlike most animal phosphodiesterases,
whose pH Optima lie on the alkaline side of neutrality,
the carrot leaf phosphodiesterase system exhibited a pH
<optimum similar to plant acid phosphomonoesterases, i.e.,
aapproximately 5.0. Acetate buffered at pH 5.2 was used

for most of the further studies.

Effect of Mgz+, Mn2+ and F”

The activity of cyclic phosphodiesterases from
eanimal tissue is stimulated by Mg2+. The leaf system also
sshowed an increased activity (65%) when Mg2+ (0.4 mM)

\vas present in the reaction mixture (Table 8). Mn2+ did
rust cause an increase in activity when present at the same
(xancentration (0.4 mM). In fact, if anything, it appeared
tflnat Mn2+ actually inhibited the system. Apparently,

Nh12+ cannot replace Mg2+ in the leaf system, at least

not at the concentration used in this investigation. The

92

50“

401

mu moles Pi/hr

20*

10*

 

Figure 26. Effect of pH on phosphodiesterase
activity.

93

 

mm Ass o.mc m +

 

mm ASE a co +msz +
. 0
mos ASE a co +m 2 +
cos amass namm OZIIHOHueoo
mua>flpo¢ m>aumamm usmaummua

 

.mz¢0 Scum oomwmamu uncommonm mop wo soavmsflfinmumc on» How
com: mums whommm osmosmum .oHMm ESHcOm muH Eosm omnmomnm mma um amuamm mpmuoum
mnu Eoum omummmum muoz +mc2 com +mmz .m.m mm .mpmpmom 2 oa.o CH cmummmnm mumz

muamm Has .mufl>fluom ommuopmmflcocmmozm so m can .+ch .+mmz mo powmwm:l.m mqmse

94

presence of F- (6.6 mM) had an inhibitory effect on

the release of phosphate from cAMP.

Identification of Reaction
Products

Evidence was obtained that 3'-AMP was the major
.immediate product formed by the hydrolysis of cAMP. This
vvas realized by paper chromatography of a reaction mixture
:in which fructose 1:6 diphosphate (2 umoles) was included
eat a relatively high concentration in order to "protect"

tzhe nucleotide product from non-specific phosphomonoester-

aase activity. Traces of 5'-AMP were also detected on the

c:hromatograms. The product of the nucleotidase, in
addition to Pi, was determined to be adenosine. Elution
cxf these compounds from the chromatograms and rechromatog-
rtiphy by thin layer technique showed them to be indis-

tignguishable from the commercial compounds co-

chromatographed with them.

Corroboration of these results was obtained when
FTHllard (personal communication) demonstrated that
incanbation of l4C-cAMP with the preparation in the presence
Of 'unlabelled 3'- and 5'-AMP gave rise to label in both
morloesters .

Sutherland and Rall (46) reported that 5'-AMP was
the: only product formed from the hydrolysis of cAMP by
the: partially purified beef heart preparation. The same

iSOuner has been found to be produced by the diesterases

95

from other animal sources. In this respect, then, the

higher plant enzyme is quite different from the enzymes

found in animal tissue.

Sucrose Density Gradient Analysis

The sedimentation profile on sucrose gradients
(of enzymes effecting release of Pi from 3',5'-cAMP showed
‘the presence of two major bands of activity (Figure 27).
VVhen 2',3'-cAMP was used, at least three bands which
llydrolyzed this substrate were determined to be present.

A comparison of the two sedimentation profiles
asuggested that different kinds of enzymes might be present
inn the preparation which were capable of hydrolyzing the
tnvo substrates. Thus, the fastest sedimenting band
agqparently represented a non-specific enzyme which could
h§ndrolyze both the 2',3'- and the 3',5'-phosphodiester
linkages. However, the slower sedimenting band catalyzing

thee hydrolysis of 3',5'-cAMP was not congruent with either

Of‘ the two slower sedimenting forms that hydrolyzed

2. (3'-CAMPI

Michaelis Constants

The apparent Km for the leaf phosphodiesterase

SYEVtem with 3',5'-cAMP as substrate was determined from a

dfinllole reciprocal plot (35), as shown in Figure 28. A

val—lie of 0.91 mM was calculated. This value is somewhat

hiérller than most constants reported for the enzymes from

96

 

 

 

l
‘7

Fraction Number

Figuxna 27. Ultracentrifugation patterns of phosphodiesterase
aetififlity with 3',5'-cAMP and 2',3'-cAMP as substrate. Protein
(15 Hug) was layered on sucrOSe gradients (5-20%) and centrié
fugeéi at 30,500 rpm for 22 hours at 4 C. Fractions collected
were assayed for activity toward'3' , 5-cAMP (o) and 2',

3 ‘CAHMP (t3). Assay conditions were as desoribed in Materials
and Methods.

 

97

 

 

 

 

 

10..

3..
E 6‘
O
m
1‘
Q
Q
a 4.

1.45101
2 app.Km = 3.33mM
O . : e :
1 2 3 4 5
[31-1 (m‘1 x 10‘3)

Flgure 28. Comparison of theMichaelis constants for 3'
nucleotidase and cyclic nucleotide phosphodiesterase activities.
variable concentrations of 3'-AMP, 2',3'-cAMP and 3',5'—cAMP
were uSe‘d for thedetermination of Michaelis constants-

Other than theconcentration of phosphate ester, all conditions
were.as described in the standard procedures given for nOn-
:KClic and cyclic substrates. Indicated in the figure are

(De apparent K values calculated for3'-AMP (A), 2' ,3'-cAMP
) and 3',5'-CAMP (0).

98

aanimal sources but it is less than the 2 mM value given

fkar the enzyme found in Dictyostelium (53).

 

Michaelis constants were also determined for the
Stibstrates 2',3'-cAMP and 3'-AMP. Values of 1.45 mM

a11d.3.33 mM were obtained respectively.

 

DISCUSSION

It has been demonstrated here that the crude
preparation from carrot leaves is capable of hydrolyzing
a large number of phosphate esters to inorganic phosphate
(Table 6). The only tested substrate that was resistant
to hydrolysis was Vitamin B12. This is not unexpected
since higher plants are known to be a poor source of this
Vitamin. Consequently, this preparation may be of value
in characterizing phosphate esters that are discovered in
the future as it may be a crude source for certain enzymes
that may be further purified for kinetic studies,
mechanism of action, etc. The list of possible phosphate
substrates is far from being complete, however, as the
results in Table 6 indicate, besides catalyzing the
hydrolysis of substrates containing a simple phosphate
ester bond, the preparation can also hydrolyze phosphate
compounds containing pyrophosphate bonds (e.g., Thiamine
pYfOphosphate) as well as high energy phosphate esters
of enols (e.g., phosphoenolpyruvate). In addition, the
phC’sphodiester linkage found in Bis-pNPP (Table 6) and
that Of cyclic nucleotide monophosphates (Table 7) were
also hydrolyzed by enzymes present in the crude prepara-

thn- It would be interesting to determine if other

99

 

100

kinds of phosphate esters, for example, oligonucleotides,
can also be hydrolyzed by the preparation.

The participation of 3',5'-cAMP in the hormonal

control mechanisms of a number of animal systems has been

firmly established (44). Only recently has the involve-

ment of this compound in a hormonal system in higher plants

been suggested. Thus, Pollard (59) has demonstrated

that exogenously applied gibberellic acid (GA), a naturally

occurring plant hormone, increases the amount of cAMP

formed from adenine-8-14C in barley aleurone layers by as

much as twice the amount formed in non-GA treated layers.
This result would be expected if cAMP was indeed partici-
Pating as a second messenger in response to the hormone,
GA- Although the synthesis of cAMP was demonstrated,
isolation of adenyl cyclase was not accomplished.

A second piece of evidence supporting the involve-
ment of CAMP in the aleurone layers was the fact that
CAMP could mimic certain of the effects caused by GA on
the synthesis and secretion of soluble sugar and other
metabolites from the layers (60). The secretion of
amYlase could also be effected by cAMP after amylase
formation was induced by suboptimal concentrations of GA.

If cAMP is active as a second messenger in higher
plants, as the above studies would suggest, then a mechanism
for CAMP degradation should be present. The results pre-

sented in this study confirmed the fact that cAMP can be

101

enzymatically degraded by a preparation. from a higher

plant source. The system studied actually hydrolyzed all

3 ' ,5'-cyclic nucleotide monophosphates tested (Table 7).
Enzyme preparations from animal systems show a preference
for cyclic nucleotides with a purine base (52).
Additionally cyclic dibutyryl AMP has been found to

be resistant to hydrolysis by phosphodiesterases prepared

from various sources (44, 52). In the carrot preparation,

however, cyclic dibutyryl AMP was actually hydrolyzed to
a greater extent than cAMP. Apparently, the plant
preparation possesses a lesser degree of specificity in
regard to hydrolyzable substrates than the animal enzymes.
The sedimentation profiles obtained from sucrose density

gradients (Figure 27) suggest that both specific and non-

specific phosphodiesterases may be present in the prepara-

tion. It is not known if these enzymes involved in the

degradation of cyclic nucleotides can also hydrolyze

Other phosphodiester compounds.

The preparation should be further purified so
that the exact specificity of the enzyme can be determined

and the assignment of a truly descriptive name for the

enzyrne can be made. In the present study, an attempt was

made to purify the diesterase by DEAF-cellulose column

chromatography under the conditions used for phospho-

trarlsferase purification (Figure 1). This resulted in

Complete loss of phosphodiesterase activity even though

 

102

the nucleotidase activity (3' and 5') eluted in a broad
enough band so that some nucleotidase activity was
present in almost every fraction of the collected eluant.
The properties of the preparation at the present
stage of purification differed markedly from most prepara—
tions obtained from other sources. Of major significance
is the fact that 3'-AMP rather than 5'-AMP is apparently
the major nucleotide product formed from cAMP degradation.
However, some 5'-AMP was also observed to be formed during
tfluadegradation of cAMP. It is not known if the same or
different enzymes gave rise to the two mononucleotides
.Another difference noted in the carrot preparation was
the acidic pH optimum (Figure 26) as compared to the
alkaline optimum noted for the enzyme from animal systems

and the enzyme found in Dictyostelium (53). The apparent

 

Km (Figure 28) determined for the carrot preparation was
found to be an intermediate value when a comparison
between the animal and fungal sources was made. In one
respect, the carrot system behaved as other phosphodi-

2+ stimulates the degradation

esterase systems in that Mg
of cAMP (Table 8).

In addition to the suggestions for further studies
alluded to above, the following investigations are also
worth pursuing: (a) distribution of the enzyme in the

cell, i.e., particulate, soluble or both? and (b) isolation

and purification of adenyl cyclase.

 

L ITERA‘I‘URE C ITED

103

 

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