THE MECHANISMS OF ARSENATE-ACTIVATION IN
ENZYMATIC REACTIONS
DISSERTATION
Presented in Partial Fulfillment of the Requirements
for the Degree Doctor of Philosophy in the
Graduate School of The Ohio State
University
By
DONALD HILLMAN SLOCUM, B. S., M. S
******
The Ohio State University
1958
Approved by
Adviser
Department of Agricultural
Biochemistry
PREFACE
Happy is the man that findeth wisdom, and the man
that getteth understanding.
For the merchandise of it
is feetter than the merchandise of silver, and the gain
thereof than fine gold.
Proverbs, 3s 13 "• I1*
ii
ACKNOWLEDGMENTS
The author wishes to express his gratitude and
appreciation to Dr. Joseph E. Varner, whose guidance
and patience were invaluable in the completion of this
work, to Dr. George C. Webster for his counsel on phases
of this problem, and to Mrs. June M. Slocum for her
editorial critique during the preparation of this
manuscript.
iii
TABLE OF CONTENTS
Page
INTRODUCTI ON
REVIEW OF LITERATURE
1
*. .......... ............
METHODS AND MATERIALS..............
3
9
I.
Enzyme Isolations
9
II.
Reaction Requirements
III.
Isolations for 0xygen-l8 Measurements...,,,
11
IV.
Synthesis of Intermediates.
13
............... 9
„
ABBREVIATIONS......................
16
EXPERIMENTAL RESULTS
17
..........
I.
Homogeneity of Glutamine Synthetase.
17
II.
Oxygen-18 Exchange in the Glutamine
Synthetase Reaction...........
17
Oxygen-18 Exchange in the Arsenolysis
of Glycogen by Muscle Phosphorylase.....
21
Oxygen-18 Exchange in the ArsenateActivated Hydrolysis of Citrulline by
Ornithine Carbamyl Transferase
24
Oxygen-18 Exchange in the Arsenolysis
of Acetyl Phosphate by Phosphoglyceraldehyde Dehydrogenase
.......
24
Oxygen-18 Exchange In the Fumarase
Catalyzed Reaction Producing Malate
26
Arsenolysis and Phosphorolysis of
Potassium Cyanate.........
29
III.
IV.
V.
VI.
VII.
VIII. Identification of Acetyl Arsenate.......
IX.
X.
36
Effect of Arsenate and Phosphate
Upon Urease Activity
............
39
Examination of Urease Activity..........
42
iv
TABLE OP CONTENTS (continued)
Page
XI.
Oxygen-18 Exchange during Decomposition
of Urea in the Presence of Arsenate and
Phosphate..........
46
DISCUSSION.....................
49
SUMMARY.........................................
68
BIBLIOGRAPHY..............
70
v
LIST OP TABLES
Page
I.
Glutamylhydroxamate Synthesis by Analytical
Ultracentrifuge Preparations of Glutamine
Synthetase....................
20
Transfer of 0xygen-l8 during the Phosphorolysis and Arsenolysis of Glutamine............
22
Transfer of 0xygen-l8 during the Arsenolytic
Degradation of Glycogen
..............
23
Transfer of Oxygen-18 in the Arsenolysis of
Citrulline.........
23
Transfer of Oxygen-18 during the Arsenolysis
of Acetyl Phosphate............
27
Transfer of Oxygen-18 during the Hydration
of Fumarate............
28
Concentration Dependence in the Arsenolysis
and Phosphorolysis of Potassium Cyanate......
30
VIII. Decomposition of Carbamyl Phosphate in the
Presence of Arsenate and Phosphate...........
33
II.
III.
IV.
V.
VI.
VII.
IX.
Effect of the Addition of High Concentration
of Anion to Depleted Reaction Mixture
34
X.
Arsenolytic Breakdown of Citrulline...........
35
XI.
Identification of Acetyl Arsenate............
37
XII.
Determination of Carbamate Formation in
Urea Breakdown by Urease
........
40
XIII. Carbon Dioxide Production In the Urease
Degradation of Urea...........
41
XIV.
Hydrolysis of Carbamyl Phosphate by Urease
43
XV.
Hydrolysis of Citrulline by Urease............
44
XVI.
Exchange of 0xygen-l8 from Arsenate and
Phosphate during the Urease Decomposition
of Urea
.................
47
vi
LIST OP FIGURES
Page
1.
2.
3.
4.
Photographs of the electrophoretic pattern of
glutamine synthetase at pH 7.4 in Tris buffer
with 0.1 ionic strength.............
18
Photographs of the ultracentrifuge pattern of
glutamine synthetase in water at pH 7.1 with
a protein concentration of 1.1 per cent........
19
Dependence of phosphate and arsenate concen­
tration upon carbon dioxide production in
the reaction of the anion with potassium
cyanate ...........................
32
Infrared spectra of acetyl phosphate and
acetyl arsenate......
38
vii
INTRODUCTION
Compounds of arsenic have long been recognized as
lethal.
Among the various inorganic and organic mole­
cules which contain arsenic, the effect of the arsenate
anion appears to be the most clearly defined.
Because
of its similarity in structure and reactivity to phosphate,
a critical anion to normal life and function, its dele­
terious effects upon phosphate metabolism are not very
surprising.
Hypotheses have been presented to account for the
effects upon enzymatic phosphorolysis•
The generally ac­
cepted view is that arsenate replaces phosphate in the
formation of essential phosphate esters.
Because of the
instability of the resulting arsenate homologs, the
normal metabolic sequence of reactions is broken.
The
end product may often be inactive toward further metab­
olism in the non-esterified or unnatural form.
An
important criterion of this postulate is the formation
of labile arsenate esters or anhydrides.
Despite the
fleeting formation of phosphate esters in some cases,
the analogous arsenates may be similarly involved in the
enzymatically catalyzed transformation.
It is proposed in this work that the arsenate esters,
1
either free or enzyme bound., are formed during arsenoly­
tic degradation of many compounds and that these esters
can he synthesized and characterizedc It has also been
proposed that there is a universality of mechanism in the
arsenate-activated hydrolyses.
Among the enzyme-substrate systems studied are
glutamine synthetase-glutamine, muscle phosphorylaseglycogen, ornithine carbamyl transferase-citrulline, and
phosphoglyceraldehyde dehydrogenase-acetyl phosphate.
The compounds synthesized during this study are
carbamyl arsenate, mono- and tri-acetyl and mono- and tri­
benzoyl arsenates.
Allied experiments carried out -with special emphasis
on their relation to the arsenolytic reactions mentioned
above include the arsenolysis of potassium cyanate and the
effect of arsenate upon the action of urease and fumarase.
2
REVIEW OP THE LITERATURE
In 1932 Harden wrote, "The close analogy which exists
between the chemical functions of phosphorus and arsenic
lends some interest to the examination of the effect of
arsenate upon yeast juice" (1).
It had been shown by
Harden and Young (2, 3) that arsenate produced considerable
acceleration of the fermentation process in yeast extract.
This alteration in rate was maintained for a considerable
period and was independent of arsenate concentration.
They
also reported that no organic arsenate esters corresponding
to the hexosephosphates appeared to be formed.
It has been
suggested that arsenate esters are formed in small quantity
and are rapidly hydrolized; therefore isolation is not
possible.
At that time, Harden mentioned that though total
fermentation with arsenate exceeds that with phosphate, fer­
mentation requires phosphate and cannot proceed in the
presence of arsenate in the total absence of phosphate.
Since Harden and Young (2) discovered the amazingly
enormous increase in the fermentation of hexose-diphosphate
by arsenate, the subject has frequently attracted investi­
gators to examine the steps of the fermentation chain for
possible site of activity of arsenate.
Braunstein, in a
series of papers from 1931 to 193^ (*J-> 5> 6, 7* 8, 9),
studied the effect of arsenate on glycolyzing erythrocytes.
Among the interesting findings were the evidence for bound
3
arsenate in an acid labile form, the parallelism of vana­
date to arsenate in stimulatory action and the preparation
of a fructose arsenate compound of possible polymeric
character*
He was in accord with the proposals of Harden
(1 ) regarding the possible existence of the labile hexosearsenate intermediates.
The first advance concerning the
specific effects of arsenate was made by Meyerhof (10) and
Meyerhof and Kiessling (11) who found that the primary
action in glycolysis and fermentation occurs during the
transformation of triose phosphate to phosphoglyceric acid.
It was shown later that phosphoglyceraldehyde is oxidized
in the presence of phosphate with subsequent production of
ATP (12, 13, 1^).
However, when arsenate is substituted
for phosphate, the oxidation proceeds with equal speed while
in the presence of both anions, the rate of oxidation is
unchanged and the phosphate uptake is reduced to zero.
Warburg and Christian (15, 16) and Negelein and Brorael
(1 7 , 1 8 ) showed that the reversible stoiciometric coupling
reaction forming ATP could be traced to a 1, 3-diphosphoglyeerie acid intermediate.
Warburg and Christian (16)
proposed that the observed effect of arsenate was due to the
spontaneous decomposition of the 1-arseno compound.
They
theorized that the hydrolysis proceeds because of the ar­
senate instability in contrast to the relative stability of
the phosphate counterparts.
This explanation, though lack­
ing any direct experimental evidence, may be accepted as
very probable (19).
Doudoroff et al. (20) extended this
concept when they found that glucose-l-phosphate was de­
graded in the presence of arsenate and the enzyme, sucrose
phosphorylase.
The product is glucose and no arsenate
ester accumulates.
Sucrose underwent the same “arsenolytic
decomposition", a term introduced by these researchers.
Similar work was done with potato phosphorylase (21) and
with muscle phosphorylase (22).
Following these reports
was the investigation of the arsenate-activated decompo­
sition of acetyl phosphate (23, 24).
The enzyme was pre­
pared from Clostridium kluyveri and required Co A.
Harting
(2 5 , 2 6 , 2 7 ) later showed that phosphoglyceraldehyde de­
hydrogenase also catalyzed this reaction.
In 1952, Khivett
presented evidence of a citrulline degrading enzyme which
functioned in the presence of arsenate or phosphate (2 8 ).
This led to an avalanche of similar reports identifying
the products as ornithine, carbon dioxide and ammonia
(29 - *10).
ylase.
Ratner (4l) named the enzyme citrulline phophor-
Study of the mechanism by Stulberg and Boyer (42)
indicated that tft,
from phosphate.
gen of carbon dioxide arises in part
Tuxs is due to an oxygen-18 exchange in a
transient intermediate.
Recently Reicbard (43) success­
fully isolated the enzyme of Grisolia and Cohen (44) which
catalyzes the carbamyl addition to ornithine.
He has shown
this to be identical to citrulline phosphorylase.
5
The arsenate-activated hydrolysis of glutamine has
also been reported (45 - 48).
The phosphorolysis has been
investigated by Boyer (49) and Ko-walski et al. (50).
They
have shown that the oxygen of the carboxyl group of glutamic
acid appears in the phosphate.
The chemistry of the production of arsenate compounds
is extensive (5 1 ) and the list of unreported compounds that
have been prepared is probably equally large.
Acyl arsen­
ates, like their phosphate counterparts, are not common; in
fact, they have never been prepared.
Crafts (52) prepared
alkyl arsenates while Wolffenstein reported the synthesis
of several mixed arsenates (53).
Some acyl phosphates have
been prepared, such as carbamyl phosphate (54), acetyl
phosphate (5 5 > 5 6 ), benzoyl phosphate (5 7 ) and mixed alkyl
acyl arsenates (55* 58).
Pictet and Bon have reported on
the preparation of acetyl arsenite and benzoyl arsenite,
giving their physical characteristics (5 9 ).
The reports that led to the study of urease have been
outlined by Sumner (60).
It was Sumner who prepared urease
as the first crystalline enzyme (6 l).
The mechanism of urea
degradation was first reported by Yamasaki in 1920 to yield
carbamic acid and ammonia (62).
Sumner et al. (63, 64) have
extended this work, confirming carbamate production in the
absence of buffer.
When neutral phosphate was added, no
free carbamate was detected.
6
The unusual characteristic of fumarasewhich renders
it amenable to the present theory of arsenolysis is the
great increase in the formation of malate catalyzed by
arsenate
(65). Fumarase is the enzyme involved in the
reaction of fumarate to malate (6 6 ).
shown by Clutterbuck
It has also been
(67) that fumarase is markedly acti­
vated by phosphate, and Massey has confirmed this (6 5 )0
Though the implications of arsenate action can be
derived from the preceding review, there are several other
reports worthy of mention for their relationship to this
problem.
Lipmann has described the arsenolysis of the
pyruvate oxidase system (6 8 ).
Crane and Lipmann have also
shown the effect of arsenate upon oxidative phosphorylation
in mitochondria
(69). Bonner (70 ) has shown growth inhibi­
tion without respiratory effects in avena using arsenate.
Maltose phosphorylase, yeast phosphorylase and other poly­
saccharide phosphorylases might be expected to behave in a
manner analogous to the phosphorylases previously described.
The enzyme described by Black and Graywhich synthesizes
beta- aspartyl semialdehyde warrents further study in re­
spect to arsenate effects
action.
(71 ). It parallels PGAD in its
The formation of a high energy phosphate in the
reaction mediated by succinyl Co A transphosphorylase may
proceed through a succinyl phosphate (72).
effect is possible.
An arsenate
The nucleotide phosphorylase of Ochoa
(7 3 ) should lend itself to similar arsenolytic studies.
The nucleosidases catalyze the phosphorolytic cleavage of
purine nucleosides to the base and the phosphopentose (7*0 •
This reaction is enhanced by phosphate which undoutedly
represents a requirement and is highly accelerated by
arsenate (75» 76).
Klein had shown that in the presence
of arsenate the free sugar was formed (77).
This indicated
a phosphate requirement and a phosphorylase activity for the
enzymes (76, 79).
The extent to which arsenate has been utilized is
enormous, but the critical use of this anion for detailed
mechanism study is, by comparison, very limited.
The realm
into which the work reported here may lead is not predict­
able but it sheds new light upon previously obtained results
by other investigators.
8
METHODS AND MATERIALS
I«
Enzyme Isolations
The source of glutamine synthetase is dried peas.
The procedure used is that of Varner (80, 8 l) which
sequentially utilizes an extraction with 20 per cent
ethanol, a calcium phosphate gel adsorption, an RNA pre­
cipitation, dialysis, Tiselius gel adsorption (82), and
finally dialysis and lyophilization.
Ornithine carbamyl transferase, "citrulline phosphory­
lase ” by previous nomenclature,was isolated by the method
of Reichard (43) from beef liver.
The purification was
carried out to the second ammonium sulfate fractionation
as described by Reichard,
Muscle phosphorylase, phosphoglyceraldehyde dehydro­
genase and fumarase were purchased from Worthington Bio­
chemical Corportion and urease was recrystallized from a
jack bean meal preparation of the Nutritional Biochemical
Company•
II.
Reaction Requirements
In the glutamine synthetase catalyzed reaction, the
complete system contained 1 mmole tris-(hydroxymethyl)aminomethane-HCl at pH 1 ,8 , 2 mmoles glutamine, 0.3 mmole
MgSO^, 0.6 mmole beta-mercaptoethanol, 0.1 mmole ADP, 2
mmoles phosphate or arsenate and 10 mg glutamine synthetase,
9
in a total volume of 100 ml.
Wien phosphate was the added
anion, 10 mg hexokinase and 100 rag glucose were present.
The reaction catalyzed by ornithine carbamyl trans­
ferase contained 5 mmoles citrulline, 3 mmoles arsenate or
phosphate, 2 mmoles Tris buffer at pH 7.4 and 50 mg of the
purified lyophilyzed enzyme preparation.
There was no measurement made of the phosphorolysis of
glycogen by muscle phosphorylase.
The reaction medium
contained 0.1 mmole ADP, 4 mmoles sodium-beta-glycer©phos­
phate with 3 mmoles cysteine-HCl buffer at pH 6 .8 , 10 mmoles
arsenate, 4 per cent glycogen (weight-volume) and 10 mg
muscle phosphorylase a, in a total volume of 100 ml.
The reaction studied with phosphoglyceraldehyde dehy­
drogenase was the arsenolysis of acetyl phospha'o first
described by Harting (25).
The complete reaction system
contained 1 mmole dilithium acetyl phosphate prepared by
the method of Avison (8 3 ), 5 mmoles arsenate adjusted to
pH 7 .8 , 0.1 mmole DPN and 10 mg phosphoglyceraldehyde de­
hydrogenase, in a total volume of 100 ml.
The urease reactions generally were carried out by
using aliquots of 100 mg urease in 100 ml of a 2 per cent
gum arable solution.
solution.
The urea was drawn from a 0.1 M stock
The arsenate and phosphate buffers were adjusted
to pH 5.0 and taken from 1 M stock solutions.
The dilithium
carbamyl phosphate was prepared as a 0.1 M stock solution
by the method of Jones et, al. (54).
10
Exact concentrations
are described for Individual sets of data.
The conversion of fumarate to malate was carried out
in a reaction mixture consisting of 1 mmole fumarate, 3
mmoles phosphate or arsenate adjusted to pH 7.3* and 0.1
mg fumarase, in a total volume of 10 ml.
The non-enzymatic degradations studied Including
potassium cyanate, carbamyl phosphate and the arsenates
are individually described with the pertinent data.
Ill-
Isolations for 0xygen-l8 Measurements
Arsenate and phosphate labeled with oxygen-18 were
prepared from enriched water containing approximately 1.4
per cent atom excess oxygen-18.
solved in excess enriched water.
The pentoxides were dis­
The solutions were placed
in sealed ampules and heated to 100°C for 72 hours.
The
solutions were neutralized with potassium hydroxide, evapor­
ated to dryness, redissolved in enriched water, sealed in an
ampule and incubated for periods in excess of 48 hours.
The products of each reaction studied required tech­
niques of isolation which in some cases were not previously
described.
The glutamic acid resulting “from the arsenolysis
and phosphorolysis of glutamine was isolated by the method
of Kowalsky ert al. (50).
Dehydration by heating produced
water which could be measured for oxygen-1 8 content by the
method of Cohn (84).
11
In the arsenolysis and phosphorysis of citrulline,
carbon dioxide is the product which was measured for oxygen18 content.
This gas was collected at liquid nitrogen temp­
eratures and measured directly in the mass spectrometer.
The measurement of the carbon-one oxygen of glucose
posed a minor problem.
The isolation from water conserving
the carbon-one oxygen was carried out by separating all
other components of the system from glucose.
After protein
precipitation, the nucleotide material was removed on acti­
vated charcoal.
The remaining foreign matter was removed
upon sequential treatment with cationic Dowex-50 and anionic
Dowex-2 resins.
Lyophilization and alcohol crystallization
gave free glucose.
Upon solution in known quantities of
water in a sealed tube at 100°C for 3 hours, the carbon-one
oxygen exchanges (8 3 , 8 6 ).
The water can then be measured
by the method of Cohn (84).
Isolation of acetate from the arsenolytic reaction
system degrading acetyl phosphate was performed in the fol­
lowing manner.
Barium chloride was added to the system until
no further precipitation occurred.
by treatment with charcoal.
Filtration was followed
The clarified supernatant was
treated with Dowex-50 resin leaving only acetate and chloride
Ions in solution.
Stoiciometric calcium hydroxide was dis­
solved in the solution and the calcium acetate was precipi­
tated with absolute alcohol.
Upon pyrolysis of calcium
acetate, acetone and calcium carbonate result.
12
The calcium
carbonate releases carbon dioxide upon acid treatment.
The
carbon dioxide was directly examined for oxygen- 18 content.
The hydration of fumarate was allowed to reach equilib­
rium.
After the precipitation of the protein, the phosphate
or arsenate was removed by precipitation as the barium salt.
After charcoal clarification, the supernatant was lyophilized
and dried in vacuo.
The dry material representing fumarate
and malate was separated, the malate heated to 150°C for 10
minutes and the water of dehydration collected and measured
for oxygen-18 content by the method of Cohn (84).
IV.
Synthesis of Intermediates
Several mono- and tri-acyl arsenates have been prepared.
Thorough characterization has been carried out on acetyl ar­
senate while some indicative tests have been made on the
other compounds as described.
Carbamyl arsenate was prepared by two methods.
The
first procedure was performed in chloroform by mixing equimolar quantities of arsenic acid and potassium cyanate.
mixture was stirred continually for 24 hours.
The
The solvent
phase was filtered and concentrated by evaporation until a
persistant yellow liquid remained.
The second procedure was an adaptation of the method of
Jones et al, (54) utilizing anhydrous conditions.
refluxed over sodium served as a solvent.
DIoxane,
Dry potassium
arsenate and potassium cyanate were added to the dioxane and
13
mechanically stirred for 3 hours.
The dioxane phase was
filtered and evaporated at low pressure.
The residual
solid is presumably dipotassium carbamyl phosphate.
Both preparations gave positive arsenate tests (8 7 ),
and were readily water soluble with gaseous evolution.
In
1 .0 N acid solution, carbon dioxide was released and meas­
ured manometrieally. In 1.0 N base solution, ammonia was
evolved and measured by back titrating a boric acid trap.
The CO2 /NH3 ratio was 1.12 for the liquid material and 1.26
for the solid preparation.
Mono-benxoyl arsenate was prepared in the reaction of
benzoyl chloride and mono-silver arsenate.
The compound is
slowly soluble in water and rapidly soluble in base.
The
hydrolysis products are arsenate and benzoic acid.
Tri-acetyl arsenate forms as a white precipitate out
of a cooled reaction mixture of arsenic pentoxide and boil­
ing acetic anhydride. The melting point of the petroleum
ether washed material was 87-93°C.
There are four moles of
acid per mole of compound as measured by titration from pH
4.0 to 6.0 in water solution.
Upon precipitation with barium
the residue was found to weigh 867 mg with the theoretical
weight in the order of 846 mg.
Two methods are described for the preparation of acetyl
arsenate.
The first method utilized monosilver arsenate and
acetyl chloride in anhydrous chloroform.
A viscous yellow
residue remaining after evaporation of the chloroform tested
14
positive for arsenate and showed two moles of acid upon
titration between pH 4.0 and 6.0.
The density was esti­
mated to be 1 .5 gr/ml.
The second method produced fine needles believed to
be disodium acetyl arsenate.
It was prepared in small
yield by apparent dehydration of sodium acetate and dibasic
sodium arsenate in the presence of one part concentrated
sulfuric acid and nine parts acetic anhydride.
was heated to 60°C with stirring.
needles were formed.
The mixture
Upon cooling fine
The crystals were washed with ether-
acetone and used for a critical analysis.
15
ABBREVIATIONS
AcAs
acetyl arsenate
AcP
acetyl phosphate
ADP
adenosine diphosphate
ATP
adenosine triphosphate
CAA
carbamyl arsenate
CAP
carbamyl phosphate
CoA
coenzyme A
DPN
diphosphopyridine nucleotide
PGAD
phosphoglyceraldehyde dehydrogenase
RNA
ribonucleic acid
Trls
tris-(hydroxymethyl)-aminomethane-hydrochloride
UDPG
uridine diphosphoglucose
16
EXPERIMENTAL RESULTS
I . Homogeneity of Glutamine Synthetase
A preparation of the glutamine synthetase enzyme was
made by the method of Varner (80, 81).
This preparation
was tested by electrophoresis and by ultracentrifugation
for purity and homogeneity.
The photographs in Figure 1
show a single major peak with some minor impurities leading
and trailing the peak.
It was calculated that the peak
contained a minimum of 90 per cent of the protein.
In the
examination of the preparation by ultracentrifugation,
Figure 2 , the protein again exhibited one peak with no
calculable impurities.
The sedimentation constant was
calculated (8 8 ) to be 17.5 s.
The activities were examined
in the supernatant of a run in which the peak was sedimented
and in the supernatant of a run in which the peak was not
sedimented.
The results seen in Table I show that the
activity is in the peak though some of the activity is lost
during the analysis.
These results show an apparently homo­
geneous protein preparation of glutamine synthetase.
II.
Oxygen-18 Exchange in the Glutamine Synthetase Reaction
It has been shown by Boyer et aJ. (49) and by Kowalski
et al. (5 0 ) that the carboxyl oxygen from glutamate is trans­
ferred to phosphate during glutamine synthesis.
This indi­
cates an intermediate formation of a glutamyl phosphate.
17
Figure 1.
Photographs of the electrophoretic pattern of
glutamine synthetase at pH 7.4 in Tris buffer
with 0 .1 ionic strength.
18
Figure 2,
Photographs of the ultracentrifugal pattern of glutamine
synthetase in water at pH 7.1 with a protein concentration
of 1 .1 per cent.
TABLE I
Glutamylhydroxamate Synthesis By Analytical
Ultracentrifuge Preparations of Glutamine Synthetase^
GluNHOH2
Enzyme Source
Prior to ultracentrifugation
0.41
Supernatant with partial
0 .2 7
protein sedimentation
Supernatant after complete
0 .0 5
protein sedimentation
^•Assay medium contains 45 micromoles Tris (pH 7
>
30 micromoles MgSOij., 10 micromoles beta-mercaptoethanol, 0.5 micromole ADP, 50 micromoles glutamine, 25
micromoles phosphate, and 40 micromoles hydroxylamine
per ml of the reaction mixture.
2Measured in 0.D, units by ferric hydroxamate complex
color, Lipmann and Tuttle (8 9 ).
20
The reverse reaction from glutamine to glutamate -was
run using oxygen-1 8 labeled phosphate and oxygen-1 8 labeled
arsenate.
The results are presented in Table II.
The
oxygen of phosphate and of arsenate is transferred to
glutamic acid during the phosphorolysls and arsenolysis
reactions.
These findings are consistent with the transfer
of oxygen- 18 from glutamate to phosphate during glutamine
synthesis.
Control experiments (Table II) with inactivated
enzyme show essentially no transfer of oxygen-1 8 to gluta­
mate (8 lj 9 0 ).
Ill*
Oxygen-18 Exchange in the Arsenolysis of Glycogen by
Muscle Phosphoryla.se
Cohn (92) has studied the oxygen transfer exhibited
during the phosphorolysis of glycogen.
Katz et al, (93)
have reported that the arsenolysis reaction proceeds in the
presence of muscle phosphorylase. Cori and Cohn (9*0 have
carried out more extensive studies on the character of this
effect.
The results led to the conclusion that a glucose
arsenate intermediate was formed in the reaction (2 0 ).
It can be seen that after arsenolysis has taken place,
the number one carbon of glucose is labeled with oxygen- 18
(Table III).
This demonstrates the existence of an inter­
mediate compound of glucose and arsenate (8 l).
Appropriate
control experiments were run and the difference between the
control with carrier glucose containing oxygen-1 8 and the
21
TABLE II
Transfer of Oxygen-18 During the
Phosphorolysis and Arsenolysis of Glutamine
Atom Per Cent Excess
System
Found
Theoretical
Phosphorolysis, P^-0-l8
0.066
0,066
Arsenolysis, Asi-0-l8
0.056
0.059
Carrier Glutamate + Pj_-0-l8^
0.011
0.000
Carrier Glutamate + As^-0-181
0.000
0.000
^Boiled enzyme used,
22
TABLE III
Transfer of Oxygen-18 during the
Arsenolytic Degradation of Glycogen-*-
System
Atom Per Cent Excess
Arsenolysis, A.s^-0-18
0,308
Carrier Glucose-0-l82
0.291
Carrier Glucose + H 2 O-I8
0,0^7
Arsenolysis, As^-0-l6
0.000
^Catalyzed by muscle phosphorylase.
p
cBoiled enzyme used.
23
trial in which the glucose was isolated from the arsenolytic reaction mixture containing arsenate labeled with oxygen18 represents a 4 per cent error.
IV,
Oxygen-18 Exchange in the Arsenate-Activated Hydrolyds of Citrulline by Ornithine Carbamyl Transferase
Since Stulberg and Boyer (42) reported their work con­
cerned with the oxygen-1 8 exchange during the phosphoroly­
sis of citrullinej a purification technique for the enzyme
ornithine carbamyl transferase has been developed (43).
The results of Stulberg and Boyer (42) show that
oxygen- 18 from phosphate appears in the carbon dioxide
evolved during phosphorolysis of citrulline.
This work has
been repeated under more favorable conditions and extended
to include the arsenate-activated hydrolysis.
Arsenate
labeled with oxygen-1 8 exchanges its oxygen with the carbon
dioxide produced during the course of the reaction (Table
IV).
The phosphate-oxygen exchange work of Stulberg and
Boyer (42) was confirmed.
The control reaction utilized
unlabeled arsenate and served as a base line.
These re­
sults were indicative of a proposed carbamyl arsenate in­
termediate (9 1 ).
V.
0xygen-l8 Exchange in the Arsenolysis of Acetyl Phos­
phate by Phosphoglyceraldehyde Dehydrogenase.
Harting (2 5 ) has shown the catalysis of acetyl phos­
phate degradation by phosphoglyceraldehyde dehydrogenase in
24
TABLE IV
Transfer of 0xygen-l8
in the Arsenolysis of Citrulline^
System
Atom Per Cent Excess
Phosphorolysis, Pj_-0-l8
0.133
Arsenolysis,, As^-0-18
0.218
Arsenolysis, As^-0~l6
0.000
1
Catalyzed by ornithine carbamyl transferase,
P^ fractions, Reichard (^3).
23
and
the presence of arsenate.
This revealed a possible ap­
proach to two mechanism problems.
One problem was the
reality of the acetyl transfer to arsenate and the other
was the probability of a l-arseno-3-phosphoglyceric acid
intermediate in the reaction normally associated with
PGAD (16).
In Table V, the results of the oxygen-18 exchange
from oxygen-18 labeled arsenate to acetate during the
degradation of AcP can be seen.
The controls were the
arsenolysis with unlabeled arsenate and an incubated
reaction mixture with carrier acetate, labeled arsenate
and no enzyme.
The procedure that was followed eliminated
dilution to a large degree and therefore the final atom
excess is large.
The divergence from the theoretical, ap­
proximately 10 per cent, may be attributed to the concurrent
dilution of the arsenate during the arsenolytic reaction.
VI.
Oxygen-18 Exchange in the Fumarase Catalyzed Reaction
Producing Malate
It was shown by Massey (6 5 ) that arsenate and to a
lesser extent phosphate greatly enhance the enzymic trans­
formation of fumarate to malate.
This was confirmed and a
method for examining the alpha-hydroxyl oxygen of malate
developed.
The results presented in Table VI are not in accord
with the proposition that phospho- or arseno-malates may
26
TABLE V
Transfer of Oxygen-18 during
the Arsenolysis of Acetyl Phosphate"
System
Atom Per Cent Excess
Arsenolysis, As^-0-18
Arsenolysis, As^-0-16
Pound
Theoretical
0.569
0.638
0.000
0.000
0.012
0.000
r
Carrier Acetate + As^-O-lS*
•^Catalyzed by phosphoglyceraldehyde dehydrogenase.
% o enzyme added.
27
TABLE VI
Transfer of Oxygen-18
during the Hydration of Fumarate^
System
Atom Per Cent Excess
Complete
0.000
Complete + H20-l8
0.102
Complete + P^-0-18
-0.009
Complete + As^-0-18
0.011
^Catalyzed by fumarase, pH 7*3
28
act as intermediates.
The oxygen-18 from water appears
in the malate hut no oxygen-18 derived from the anions is
detected in the malate produced.
VII.
Arsenolysis and Phosphorolysis of Potassium Cyanate
The release of carbon dioxide from solutions of po­
tassium cyanate upon the addition of phosphate or arsenate
at pH 5.0 was measured by namometry.
Table VII shows re­
sults of the dependence of the reaction upon the concentra­
tions of the reactants.
Figure 3 shows a non-linear plot
for the dependence of the initial rate of the reaction upon
the concentration of the anion.
Points at higher concen­
trations of anion fall on the extrapolated curve.
cyanate level was constant in these experiments.
The
The re­
sults in Table VIII give the relative effects of phosphate
and arsenate upon the non-enzymatic degradation of carbamyl
phosphate.
The final data to be presented concerning this
reaction series are those in Table IX.
There is represented
the addition of a high concentration of anion to a reaction
mixture which is apparently no longer producing a substan­
tial quantity of carbon dioxide.
Reproduced in Table X is
the data of Slade nt al. (3 8 ), presented for comparison of
the non-enzymatic with the enzyme catalyzed arsenolysis.
29
TABLE VII
Concentration Dependence in the
Arsenolysis and Phosphorolysis of Potassium Cyanate
Cyanate cone.
Anion and cone »
C02 evolved
uL/6 min
0.005M
0.05M AsO^
0
0 .0 1
0 .0 5
"
0
0.05
0.05
"
17
0 .1
0 .0 5
"
45
0.3
0.05
“
125
0.005
0.05 POi).
0
0 .0 1
0 .0 5
"
0
0.05
0.05
"
3
0.1
0.05
"
24
0 .3
0.05
"
78
0 .1
0 .1
0.001 AsO^
it
0.005
II
0.01
0.1
0 .0 5
0.1
0 .1 5
0.1
0 .3
0.1
22
31
32
II
45
II
80
II
132
30
TABLE VII (Continued)
Cyanate cone.
Anion and cone
COg evolved
uL/6 min
0.1M
0.001M PO],
ii
0 .0 0 5
18
0.1
0.01
18
0.1
0.05
2k
0.1
0.15
36
0.1
0.3
k5
0 .1
31
9
As j
50
c
E
(£>
c
0J
O
O
0.05
Figure 3.
0.1
0.15
A N IO N CONCENTRATION
Dependence of phosphate and arsenate concentration upon carbon dioxide pro­
duction in the reaction of the anion with potassium cyanate. Reaction was
run at pH 5.0, 3 0 °C and dC02/dt is expressed as uL/6 min.
TABLE VIII
Decomposition of Carbamyl Phosphate
in the Presence of Arsenate and Phosphate"*-
Addenda
COg evolved, uL/15 min
0.1M CAP
+ H20
19
0.1M CAP
+ 0.3M
AsOij.
26
0.1M CAP
+ 0.3M
PO^
13
1pH 5.0, 30°C.
33
TABLE IX
Effect of the Addition of High
Concentration of Anion to Depleted Reaction Mixture'
Addenda
CO2 evolved, uL/15 mln
#1. 0.1M KOCN + 0.005M AsO^
^5
#2. 0.1M KOCN + Q,005M PO4
27
After 15 minute incubation;
C02 evolved, uL/first
#1. add 0.3M AsO^
65
#2. add 0.3M POij.
26
1pH 5.0, 30°C.
3^
6
min
TABLE X
Arsenolytie Breakdown of Citrulline^-
uM AsOij.
CO^j uL/6 min
0
3
2
5
5
20
10
37
20
48
30
55
•^-Slade et al. (3 8 )
35
VIII.
Identification of Acetyl Arsenate
The crystals used in this analysis were isolated from
a reaction mixture of sodium acetate and sodium arsenate in
acetic anhydride and sulfuric acid.
A positive flame test for sodium was evidenced,, i.e.,a
bright yellow flame not visible through a blue filter.
Masked was any blue flame indicative of arsenic.
A Fiske-
Subbarow test (8 7 ) which gives a blue coloration with
phosphate and arsenate was run and the results are presented
in Table XI.
An hydroxamate test (8 9 ) was positive though
the quantitation was consistently low.
Upon addition of
AcAs to a solution of aniline a precipitate was formed.
This was isolated and the melting point determined.
compound was acetanilide.
The
A precipitate occurred upon the
addition of AcAs to dinitrophenlhydrazine.
A barium chloride precipitation was carried out.
The
arsenate was precipitated directly, filtered, dried and
weighed by standard methods.
The barium acetate was then
separated by alcohol precipitation and determined quanti­
tatively.
control.
A base hydrolyzed AcF solution was used as a
The results are presented in Table XI.
The infrared spectra of AcP and AcAs are reproduced in
Figure 4.
36
TABLE XI
Identification of Acetyl Arsenate
Flame Test
Na
Fiske-Subbar ow Test (8 7 )
O.D.
1 x
lcr4M AcAs
0.375
1 x
10-1*'M AsO^
0.385
Hydroxamate Test (8 9 )
O.D.
1 x
10"^M AcAs
0.205
1 x
10_ifM Ac20
0.510
Reaction with Aniline
M.P.
Precipitate
110-115
Acetanilide
114
Dinitrophenylhydrazine Test
(95)
ppt
Barium Chloride Precipitation
Found
Theoretical
Initial
precipitate(AcAs)
292 mg
34-5 mg
Alcohol
precipitate(AcAs)
133 mg
112 mg
Initial
precipitate (AcP)
291 mg
301 mg
Alcohol
precipitate (AcP)
121 mg
112 mg
37
100
80
PERCENT
TRANSMITTANCE
60
40
ACETYL
PHOSPHATE
20
100
80
60
40
ACETYL ARSENATE
20
WAVE LENGTH, MICRONS
Figure 4.
Infrared spectra of acetyl phosphate and acetyl arsenate. Results from the
Baird Recording Infrared Spectrophotometer in potassium bromide.
IX.
The Effect of Arsenate and Phosphate Upon Urease Ac­
tivity
Sumner (6 3 , 64) reported that the presence of phosphate
inhibited carbamate formation in the process of urea degra­
dation by urease.
This was repeated with phosphate and in
addition with arsenate using two analytical procedures
(64, 9 6 ).
The results are shown in Table XII.
This table
also reveals the relative production of ammonia in the
presence of the different buffers as seen in the third
column of Table XII.
The carbon dioxide production was measured at pH 5.0
in the presence of arsenate and phosphate buffers with
acetate buffer as the control.
Table XIII shows the extreme
Inhibition by phosphate and the partial inhibition by ar­
senate at the concentrations used.
The arsenate inhibition
is about 30 per cent and the phosphate inhibition of the
carbon dioxide production is above 95 per cent.
At pH 5.0
and pH 7 .0 less carbon dioxide can escape, but the apparent
enhancement by both phosphate and arsenate is indicative of
their role as part of a possible intermediate (Table XIII).
It was also determined that CAP gave a negative car­
bamate test by the Lewis and Burrows test (9 6 ) and gave
identical results in both the acid and base nesslerization
tests (64).
Negative results for the breakdown of CAP by
urease were evidenced in acetate, arsenate and phosphate
buffers at pH 5.0.
39
TABLE XII
Determination of Carbamate Formation in
Urea Breakdown by Urease
System-1-
No Buffer
Carbamate Formed
by Barium ppt (9 6 )
by Nesslerization (64)
initial
boiled
acid
30$ 2
70%
1 0 .43
base
7.5
Arsenate
lOOfo
O%
1 1 .6
11.5
Phosphate
100 ^
0fo
1 1 .1
1 0 .6
•^•Complete system contained 1 ml urease in 2 per cent gum
arabic solution, 100 mg urea and water or 0 .3 M arsenate
or phosphate buffer at pH J.0, in a total volume of 5 ml*
2A s
per cent of precipitate formed.
3a s mg of NH^ formed.
4o
TABLE XIII
Carbon Dioxide Production in the
Urease Degradation of Urea
System-1-
uL CO2 produced/30 min
pH 6 .0
pH 7 .1
pH 8.1
I87
34
5
0
127
91
67
27
5
64
60
27
pH 5.0
Acetate
2
Acetate and Arsenate
Acetate and Phosphate
-^-Complete system contained 1 ml urease in 2 per cent gum
arabic solution, 1 ml 0.1 M urea, 1 ml 0.1 M acetate
buffer pH 5.0 and 1 ml of 0.3 buffer.
2Total concentration of acetate buffer is 0.4 M.
X.
Examination of Urease Activity
As a result of the experiments which reveal unusual
urease activity in the presence of phosphate and arsenate,
urease activity was examined more closely.
Assuming that
some intermediate involving phosphate or arsenate may exist
and that a carbamyl intermediate, more specifically, may be
involved, the specificity of urease was examined.
pounds seemed reasonable to test.
citrulline.
Two com­
They were CAP and
It was shown by following carbon dioxide re­
lease that CAP was degraded slowly but only in the presence
of ammonium ion (Table XIV). Citrulline was also degraded,
though very slowly.
Arsenate and phosphate effected this
decomposition in a manner similar to the decomposition of
urea (Table XV). CAP degradation was very slightly enhanced
by the addition of arsenate in the presence and absence of
ammonium ion (Table XIV) .
Urea decomposition in the presence of phosphate was
run and an attempt to isolate CAP was made. A chromato­
graphic system which uses a 3:1 ratio of acetone to 25 Per
cent trichloroacetic acid separated CAP and inorganic phos­
phate on paper.
The reaction mixture yielded no CAP by
colorimetric methods but a phosphate containing spot which
moves more rapidly than phosphate was found.
A more sensi­
tive detection was allowed by utilizing phosphate labeled
with phosphorus-32.
The result was negative with all the
activity appearing in the inorganic phosphate spot.
42
TABLE XIV
Hydrolysis of Carbamyl Phosphate by Urease
Addenda
uL COg released/30 min
71
CAP
CAP +
WH]j.
30
CAP +
Asi
13
CAP +
NH4 + ASj_
36
^Corrected by using a control containing enzyme in the
flask and CAP in the side arm (unmixed).
^3
TABLE XV
Hydrolysis of Citrulline by Urease
Addenda
uL CO^ released/30 mi
no addition
2
citrulline
22
citrulline +
5
citrulline + As*
33
44
The possible exchange between CAP and inorganic phos­
phate was examined*
There was no exchange in the presence
or absence of urea.
Urease, therefore, was not capable of
catalyzing this exchamge.
Once again all of the radioactiv­
ity was found in the inorganic phosphate spot.
Another approach was to determine any net synthesis of
urea by urease from CAP in the presence of ammonia.
By
using diacetylmonoxime to detect urea, no production could
be shown•
The transferring ability of urease was also measured.
Urea was degraded ly urease in the presence of aspartate.
Chromatography revealed no definite spots which could be
recognized as ureidosuccinic acid though streaming occurred
preceding the aspatate zone in the unknown sample.
This
was not compared to a ureidosuccinic acid known.
The decomposition of citrulline was then examined
chromatographically.
The appearance of a ninhydrin positive
spot, possibly ornithine, was shown to occur.
This confirmed
the manometric data which showed a citrulline induced re­
lease of carbon dioxide.
More critical data are necessary
to advance a transfer or non-specific hydrolytic function
for urease, though some unique aspects are evidenced in
these cursory experiments.
45
XI.
Oxygen-18 Exchange During Decomposition of Urea in the
Presence of Arsenate and Phosphate.
Carbon dioxide and ammonia are the products of the
urease decomposition of urea.
The carbon dioxide production
is affected by arsenate and phosphate.
The anions may be
implicated in the reaction as acceptors of the carbamyl
moeity -which Sumner (6 3 , 64) and Yamasaki (62) propose as
one of the products of the reaction.
If this was the case,
it is anticipated that oxygen-18 exchange similar to that
experienced in the "citrulline phosphorylase" reaction
should occur.
The results of the oxygen-18 experiments are reported
in Table XVI.
The reaction mixture containing arsenate
labeled with oxygen-18 produced carbon dioxide more rapidly
than the phosphate containing medium.
The greater figure
for oxygen-18 exchange from arsenate to carbon dioxide
(Table XIV) represents the initial gas collected in the
first five minutes of reaction.
The smaller number rep­
resents carbon dioxide collected over a thirty minute inter­
val.
Both figures indicated a substantial quantity of
oxygen-18 resulting from arsenate.
The phosphate affected
reaction produced small amounts of carbon dioxide over the
thirty minute collection period.
The exchange of oxygen-18
from phosphate to carbon dioxide is readily seen.
(Table XIV)
The control experiments show essentially no oxygen-18.
release of carbon dioxide in the presence of arsenate
46
The
TABLE XVI
Exchange of Oxygen-18 from Arsenate and Phosphate during the
Urease Decomposition of Urea
System
Urea * Asj,-0-l8
Atom Per Cent Excess
0.2081
1
Urea + A sj -0-18
0.0302
Urea + P^-0-l8
0.168
Ammonium Carbonate+ As^-0-18
0.007
Urea + Acetate-0-16
0.000
-*-C02 from first five minutes of reaction.
2 C02 from 30 minute reaction interval.
^7
labeled with oxygen-18 serves as a control for the other
reactions which involve a production of carbon dioxide.
48
DISCUSSION
The experiments reported here have their primary pur­
pose in the contrivance of a theory covering the in vitro
effect of arsenate on enzymatic reactions.
That this theory
is in accord ■with the suggestions of Harden (1) and Braunstein (4 - 9) and the hypothesis of Warburg and Christian
(15) will become evident throughout this discussion.
The
premises of the existing proposals were supposition ex­
pedient and intrinsic on the basis of the available data.
With the advent of experiments from which direct evidence
can be obtained concerning the role of the arsenate anion,
a more complete evaluation is accessible.
Secondarily, though of apparently equal importance,
there is the possibility of more detailed analysis of the
mechanism of each individual enzyme catalyzed reaction
studied.
Much of the data allows speculation in the con­
sideration of the in vivo reactions.
The lack of informa­
tion on arsenate anhydrides beyond that presented in this
work limits the scope of the theory.
It is intended that
the implications of the initial findings will stimulate cal­
culated predictions, some of which are amenable to experi­
mental confirmation.
Before beginning the survey of the arsenate affected
reactions, a short discussion on enzyme purity and methods
shall be made.
Among the enzymes utilized in this project,
49
none were purified by techniques designed by the author.
Glutamine synthetase was obtained from dried peas by a
method developed by Varner (80).
The increased activity as
compared to other glutamine synthetase preparations was
known.
Since Boyer (91) had predicted that oxygen exchange
with arsenate was impractical with reported preparations,
the availability of this new method of isolation and puri­
fication allowed the extension of oxygen exchange measure­
ments to the arsenate-activated hydrolysis of glutamine.
The examination of the protein prepared by the method of
Varner (8 0 ) revealed an homogeneous protein which contained
the activity.
The assurance of the homogeneity lies in the
electrophoretic and the ultracentrifugal patterns as they
appear in Figure 1 and Figure 2 respectively.
A sedimenta­
tion constant of 1 7 .5s was calculated and the rapidity with
which the enzyme migrates in the centrifugal field indicates
a large size molecule.
This constant can be compared to the
13.9s figure reported by Levintow et al. (97).
The enzyme
preparation of ornithine carbamyl transferase was made by
the method of Reichard (^3).
Prior to his report an attempt
to purify ’’citrulline phosphorylase” was in progress in
this laboratory.
The initial results may allow an alternate
method of preparation if carried further.
Liver mitochon­
dria were prepared and represented a major purification
step.
The freezing and subsequent thawing removed more un­
desirable protein, material with little loss in activity.
Nothing was done beyond this point but the result was a
very active preparation.
In all the oxygen-18 experiments
performed, substantial quantities of purified enzyme were
required in order to complete sufficient reaction before
extensive dilution of the arsenate oxygen with water could
proceed.
When dealing with the reaction catalyzed by glutamine
synthetase, it is necessary to discuss the role of phosphate
in the synthetase reaction.
Boyer and coworkers (^9) and
Kowalsky _et al. (5 0 ) have shown that the oxygen from
glutamic acid is transferred to the phosphate from ATP
during the synthesis of glutamine.
This would indicate a
glutamyl phosphate intermediate though Levintow and Meister
(9 8 ) have reported that glutamyl phosphate does not act as
an intermediate.
Since details of this experiment are ob­
scure, careful repitition with a highly active enzyme prep­
aration is a worthy project.
The overall reaction catalyzed
by glutamine synthetase is as follows:
Glutamate + ATP + NH^ ^
Glutamine ■+ ADP + P^_
As reported by Varner et aJL. (81) the reverse exchange of
oxygen-18 from phosphate to glutamate can be shown.
In the
course of this reaction, when glutamine is deamidated,
arsenate can replace phosphate.
There are three possibilities
which exist for describing the reaction mechanism.
is the formation of a glutamyl arsenate.
The first
The second pos­
sibility is the formation of an enzyme-arsenate. Finally,
an Intermediate ADP-arsenate can be postulated.
Only one,
the former, is amenable to the exchange data and to the
arsenolysis scheme which will be discussed in detail later.
The enzyme-arsenate compound would require displacement of
the arsenate by a glutamyl group and a catalytic dilution
of the oxygen-18 of arsenate.
This mechanism has been
covered for phosphate elsewhere (50).
An ADP-arsenate in­
termediate can account for the labeling of glutamic acid
but only through an unwieldy mechanism.
Effectively, there
is little that can be summized from these data concerning
the mechanism of action of glutamine synthesis.
The phos-
phorolysis confirms and strengthens the proposition pre­
sented by other workers (49, 50).
The arsenolysis proceeds
in an identical manner and by analogy should form a glutamyl
arsenate Intermediate.
Since this Is the case, one might
suspect a glutamyl transfer from ammonia to the anion fol­
lowed by a subsequent transfer of the anion to a nucleotide
acceptor.
These steps would be catalyzed by this enzyme an
the glutamyl radical as well as the glutamyl-anion anhydrid
could be enzyme bound.
In the situation in which arsenate
is the anion utilized, a transfer of the anion to the
nucleotide may not be consummated.
An hydrolysis of the
postulated glutamyl arsenate, perhaps enzyme induced, re­
sults in the recognized arsenolysis reaction.
The second enzyme studied was muscle phosphorylase.
Cohn (99) had shown that in the phosphorolysis of glycogen
in the presence of phosphate labeled with oxygen-18, the
oxygen of the hemiacetal bond was labeled.
The bond fission
was shown to be dependent upon conditions of hydrolysis
though more frequently the carbon to oxygen bond was broken.
Considering this, the arsenolysis should form a transient
glucose-1-arsenate and the probability of finding oxygen-18
in glucose becomes dependent upon the type of fission.
These results show that the glucose-1-oxygen is labeled in­
dicating an intimate relation of glucose and arsenate and,
assuming a glucose-1-arsenate intermediate, an acyl fission,
i.e. the breaking of the oxygen to arsenic bond, takes place.
The acyl fission could have been anticipated on the basis
of the relative instability of the oxygen to arsenic bond
revealed by the ease with which arsenate oxygens exchange
with water.
Another suggestion concerning the weakness of
the oxygen to arsenic linkage would be derived from the
knowledge that arsenate is more basic in character than
phosphate.
The antimonate anion which exists as Sb(OH)g
(100) is basic in nature and forms unusual salts in which
hydroxyl groups are replaced (101).
Finally the tendency
of arsenate to be reduced to arsenite shows relative in­
stability of the oxygen to arsenic linkage as compared to
phosphate.
The probable formation of a glucose-l-arsenate
has not been demonstrated and the proposal of Doudoroff
jet al. (20) confirmed by direct experimental evidence.
The isolation of glucose for the determination of oxygen-18
did not follow the procedure of Koshland and Stein (102)
but was a more simplified method devised in this labora­
tory.
The effect of arsenate and phosphate upon citrulline
metabolism has been frequently studied but a side from the
report <f Krebs and coworkers (103), the mechanism has been
neglected.
Reichard (43), by isolating and purifying the
citrulline synthesizing enzyme and characterizing its
activity, has enabled researchers to direct their thinking
toward more specific mechanisms.
Since citrulline phos-
phorolysis results in the production of ornithine and CAP,
it is anticipated that the oxygen-18 of phosphate would
appear in the carbon dioxide from decomposing CAP.
This
was shown by Stuiberg and Boyer (42) and confirmed in this
laboratory, Table IV, under more favorable conditions of
enzyme purity and of collection technique.
As Reichard
(43) pointed out and Glasziou (104) had shown, CAP was
involved in a carbamyl adenosine diphosphate phosphoferase
reaction in which carbon dioxide and ATP are among the
products.
When, arsenate replaced phosphate, carbon dioxide
was produced and this reaction proceeded in the presence of
pure ornithine carbamyl transferase.
It is not unreasonable
to assume a CAA intermediate, which according to present
theory, spontaneously decomposes to yield carbon dioxide.
This has been subjected to an oxygen-18 exchange experiment
similar to that described for phosphorolysis. The results
54
(Table IV) demonstrated that CAA, may Indeed have been
formed.
The reactions involved are those previously de­
scribed except reaction (C) below, which is an inclusive
activity of the ornithine carbamyl transferase enzyme.
Reaction (D) is proposed to be enzymatically catalyzed
by the transferase.
(A)
NHgCOOH + ATP
GAP 4- ADP
(B)
CAP 4- Ornithine
(C)
Citrulline 4- As^
Citrulline + P^
CAA 4- Ornithine
(D)--CAA --- >
C02 + NH3 4- As±
(E)
C02 4-NH3 4- Pi
CAP
>
The first reaction, (A),is catalyzed by theenzyme de­
scribed by Glasziou(104).
The fifth reaction, (E), must
be proposed on the basis of nonenzymatic data.
This arises
from the kinetics of the reaction descried by Jones (5^4)
for the preparation of CAP.
When cyan. ,e and phosphate are
reacted at 30°C the carbon dioxide production at low anion
concentration is dependent upon the first order concentra­
tions of the reactants.
At increased concentrations of
anion, the curve is a straight line with a decreased slope.
The implication of this is that as CAP is formed at low
phosphate concentration, the rate of decomposition is
directly proportional to CAP formation.
When phosphate
concentration is increased, the rate limiting step is the
decomposition of CAP which is proportional to the reactants
but possibly with a new set of constants.
55
It Is assumed in
one instance that the irreversibility of the second order
step of the reaction is a result of an inhibition by the
relatively large concentration of anion.
It may be that
all the cyanate is reacted and occurs as CAP.
In either
case there is an accumulation of the CAP since it is iso­
lated in a 70 per cent yield.
The reaction of arsenate plus
cyanate follows the same pattern though the carbon dioxide
is produced about three to four times as rapidly.
The total
production of carbon dioxide in thirty minutes represents
about 10 per cent of the theoretical from cyanate.
It is
therefore concluded that either CAA is accumulating or
there is a truly unique difference in the reactions of
cyanate and phosphate and cyanate and arsenate. In order
to have such striking similarity In the rate versus concen­
tration curves, any difference must be proportionally com­
pensated by a second divergence.
This is difficult to
project into the actual process and it seems rather un­
likely.
If CAA is accumulating during the reaction, an
isolation could be made though it is technically difficult.
Herein lies the first new stipulation which must be recog­
nized in the arsenolysis reactions.
to have a substantial half life.
The CAA compound seems
Therefore, it must be
considered as a possible substrate for other reactions.
This shall be covered in more detail later in the discussion.
In the examination of the mechanism of arsenolysis it
seemed necessary that the classical Warburg and Christian
56
(1 5 ) reaction should he studied using oxygen-18 labeled
arsenate.
Since Harting (25) had shown that PGAD was
capable of the reduction of acetaldehyde yielding acetyl
phosphate, that arsenate could replace phosphate in this
reaction and that arsenate could also effect the arsenolysis
of acetyl phosphate, this simplified tool was utilized.
Upon the addition of arsenate labeled with oxygen-18 to the
AcP and PGAD mixture, acetate was formed.
The carboxyl
oxygens were liberated as carbon dioxide and measurements
showed oxygen-18 to be present (Table V). These results are
in accord with the formation of acetyl arsenate.
Provided
with a satisfactory assay system, it would be of interest
to compare the nonenzymatic hydrolysis of acetyl arsenate
with the PGAD induced hydrolysis.
The possibility that the enhancement of fumarase ac­
tivity by phosphate and arsenate might be due to an alphaester intermediate led to the examination of this reaction
with oxygen-18 labeled anions.
The negative results
(Table VI) appear to rule out this mechanism.
Prom these data, a composite theory of the mechanism
of arsenolysis can be proposed.
Warburg and Christian (15)
presented the hypothesis that a l-arseno-3-phosphoglyceric
acid intermediate was formed and being labile spontaneously
decomposed to form 3-phosphoglyceric acid (17, 18).
Doudoroff et al. (20) put forth the proposition that the
arsenolysis of sucrose proceed through a labile glucose-157
arsenate ester.
Therefore, based upon the data presented
here, the first consideration in arsenolysis reactions is
that such intermediates are formed and are labile in so
far as the isolation techniques and assay methods are con­
cerned.
Several other facets must be reviewed.
It has
been shown that yeast can tolerate extremely high levels
of arsenate and survive (105, 106).
In fact microbes
growing on low phosphate and not adapted to arsenate res­
pond more rapidly to arsenate than to phosphate (up to
specific concentration levels). This growth increase was
substantial (107).
Though there are several possible ex­
planations, it is necessary that the actual physiological
replacement of phosphate by arsenate to some degree must
be considered.
The indications are that in water solution,
arsenate anhydrides have a reasonable half life.
If this
is the case, the extreme lability of these intermediates
must be explained.
The lability may be in the isolation
techniques as was mentioned previously or the intermediates
may never appear in a free form.
However the hydrolysis
may occur upon the enzyme surface as an enzyme catalyzed
hydrolysis.
This proposal would account for several ar-
senolytic discrepancies.
These divergencies are:
1)
Arsenolysis proceeds more slowly than phosphorolysis in
some instances.
2)
Arsenate enhances transfer and does
not produce hydrolysis in some cases.
3)
Some products
of phosphorolysis are arsenolyzed while others resist this
58
action.
4)
Arsenate replaces, in part, phosphate in vivo.
The first issue could have two answers.
First, the rate of
transfer to arsenate is slower than to phosphate in the few
enzymes such as muxcle phosphorylase. If this were the case,
small quantities of phosphate would substantially annul
arsenolysis.
It is known that glucose-1-phosphate in the
presence of glycogen is arsenolyzed as rapidly as glycogen
which is in opposition to the acceptor proposal.
Sufficient
phosphate would become available in the course of the reac­
tion to overwhelm the arsenate effect.
Therefore, a de­
crease in rate and in total arsenolysis should have been
noted.
The second approach is that an enzymatic hydrolysis
is the rate limiting step and the enzyme bound glucose-1 arsenate is relatively stable toward hydrolysis.
This
assumes that arsenate intermediates are seldom released
from the surface of the enzyme.
For enzymes which show
equal or greater rates of arsenolysis as compared to phos­
phorolysis, it can be summarized that either the phosphate
ester products are not removed rapidly enough during phos­
phorolysis or that the hydrolysis of the arsenate esters on
the enzyme is swift.
To question two, it can be answered that the addition
of a more preferred compound than water can be made to the
arsenate-ester-enzyme complex more readily than to a cor­
responding phosphate complex.
enzyme induced hydrolysis.
This is equivalent to an
Here if free compounds of the
59
anions existed, the present theory would require organoarsenate hydrolysis to occur spontaneously.
For this
reason, an enzymatic catalysis Is preferred to the pro­
posal of the nonenzymatic spontaneous decomposition of
the organo-arsenate.
In regard to third point of Issue, it may he conceived
that the enzyme tends to hind the organic and inorganic
anion portions firmly and tends to lack the ability to
sever the hond in the absence of the proper acceptor.
Thi3 represents its resistance to hydrolysis and most
enzymes which exhibit this tendency have rates of arsen­
olysis slower than rates of phosphorolysis.
Since the
binding of the substrate is relatively strong, there is
no exchange of either portion of the ester except in the
presence of the physiological acceptor.
Those enzymes
that do show arsenolysis of the substrate or the product
of phosphorolysis also show phosphate or organo-exchange.
The replacement of phosphate by arsenate can partici­
pate more efficiently in specific reactions (48, 6 5 ).
It
has been argued that those reactions in which arsenolysis
is slow, the Intermediate enzyme complexes are stable toward
hydrolysis.
For this reason arsenate could enhance the
former, be ineffectual In the latter, and inhibit only in
those reactions where arsenolysis is very favorable such
as PGAD.
Even in the last instance, the possibility of
transfer can exist.
This would require either a free
60
organo-arsenate, If such exists, or an acceptor more
preferred than water.
Also, phosphate inhibition would
play a protective role for these enzymes.
A mutation or
adaptation of the hydrolytic qualities of the enzyme might
be necessary for adapted species.
these situations are as follows.
Examples for each of
The transferase activity
of glutamine synthetase is enhanced by arsenate.
The
polysaccharide phosphorylases would not be effected suf­
ficiently to alter the course of their function.
The ac­
tivity of PGAD would be Inhibited in so far as its produc­
tion of the diphosphoglyeerie acid.
Therefore, at high
concentrations of arsenate, yeast would require only muta­
tions to compensate for enzymes acting similarly to PGAD.
Arsenolysis therefore, is depicted here as entirely enzymatic
in the mode of action.
The spontaneous reaction decomposing
arsenate esters (1 5 ) is catalyzed by the phosphorolytic
enzyme.
It would be of interest to compare enzymatic ac­
tivities of specific phosphorolytic enzymes from arsenate
adapted yeast with those enzymes from normal yeast.
Adapted yeast seems to form free arsenate esters which are
acid labile and this is contrary to a spontaneous nonenzymatic hydrolysis (6 ).
It has been shown also by Carr
(107) that Azotobacter grown on minimum phosphate is en­
hanced by a 20 per cent arsenate-phosphate mixture above
an equimolar phosphate control.
Such results allow for no
mutations, therefore, rely upon functional utilization of
61
arsenate.
Though the enhancement of growth by arsenate
occurs at relatively high levels of arsenate, another ex­
planation rather than the selective replacement of phosphate
by arsenate may be made.
The enhancement caused by subin-
hibitory concentrations of inhibitors seems to be mediated
by a shift toward a more efficient balance between energy
and structural material (108).
This could result from
arsenolysis of key compounds such as acetyl-Co A which
decreases the extent of degradation.
A partial uncoupling
of oxidative phosphorylation may effect growth in a similar
manner.
These possibilities complicate definite commit­
ments on in vivo systems.
Depicting the general arsenolysis scheme requires two
reactions.
The definition of terms and the reaction
sequence are as follows:
0 n — organic compound susceptible to arsenolysis
0 n-l =■ secondary product of arsenolysis
A = arsenate anion
E =s enzyme
O-E-A = enzyme complex of a proposed organo-arsenate
0 = major product of arsenolysis
E
0 -4- A
O-E-A + 0n -i
n
ii--L
O-E-A + H20 — — > 0 + E + A
An interesting reaction which finds support in and
lends support to the proposals presented, is a rather
unique functioning of urease.
For a comparative survey
62
on urease, an outline of some of the earlier literature
presented by Sumner and Somers has been used (109).
The
most prominent facets of their review deal with specificity
and with mechanism.
For more recent aspects of ionic
effects, the report of Fasman and Niemann (110) presents an
interpretation which may make desirable a re-evaluation of
the published work on urease kinetics, ionic strength
effects and buffer and pH optima.
their conclusions.
Further has supported
The conclusion they reach is that
anions activate and cations inhibit urease and that the
net effect is the resultant of two opposing tendencies.
In the work reported here, every effort has been made to
avoid complications such as they describe.
Urease has been called "absolutely specific" (109).
Werner (111) has reported the decomposition of N-monobutyl
urea though Sumner denies the veracity of this work (109).
The similarity of N-monombutyl to citrulline by assigning
a nomenclature of an N-substituted urea to citrulline.
The significance of this shall be discussed.
The mechanism of the reaction catalyzed by urease has
been mentioned previously.
Sumner and Myrbeck (6 0 ) present
chronologically an historical outline ending with the con­
clusion that in the absence of buffers, ammonium carbamate
is formed from the products carbon dioxide and ammonia.
In the presence of buffers, carbonic acid and ammonium
salts are preferentially formed (64).
The recent work of
Wang and Tarr (112) using ’water labeled with oxygen-18
has confirmed by an elegant procedure that carbamate and
ammonia are the primary products.
Relying upon these
critical data, it may be assumed that urease is a carbamyl
transfering enzyme which transfers its carbamyl group to
water.
There is no inhibition of ammonia formation in
the presence of arsenate or phosphate while no detectable
carbamate is formed (Table XII).
The outstanding effect
of arsenate and phosphate is the alteration of the produc­
tion of carbon dioxide.
At pH 5.0, phosphate completely
inhibits the production of carbon dioxide.
This may be
explained on the basis of an enzyme-carbamyl-phosphate
complex which is only slowly hydrolyzed (Table XIV).
Perhaps the nitrogen-15 study of Roberts* group (113)
which shows an exchange of nitrogen between urea and
urease would be indicative of a covalent linkage and there­
fore confer stability on the proposed intermediate.
Arsenate exhibits tendencies similar to phosphate but the
relative instability of an enzyme-carbamyl-arsenate complex
may allow only partial inhibition of carbon dioxide pro­
duction.
The confirmatory experiments are those in which
pH was varied and those in which labeled anions were used.
As the pH is increased the release of carbon dioxide is
decreased simply by the relative solubility of carbon
dioxide.
This can be seen in Table XIII.
Two effects
rule the change in the presence of the anions.
One Is the
solubility of carbon dioxide and the second is the stability
of the enzyme complex.
Above pH y.O, the complex with ar­
senate or phosphate would be so unstable no differences
between the arsenate or the phosphate complex should be
detected.
At the same pH levels, pH 7.0, phosphate and
presumably arsenate activate urease (110).
If this pos­
tulate holds then one may anticipate finding CAP in a
urease reaction mixture to which phosphate has been added.
None could be detected either colorimetrically or by using
phosphate labeled with phosphorus-32.
A possible phosphate
exchange between CAP and inorganic phosphate could have
existed but this was shown to be negative.
labeled with phosphorus-32 was used.
Phosphate
The use of oxygen-18
in the anions yielded more fruitful results.
As it was
expected, oxygen-1 8 from the anions appeared in the carbon
dioxide released.
From the work of Wang and Tarr (112)
it may be said that such results require an intimate re­
lationship between the carbamate and the anion in the form
of a free or enzyme bound intermediate.
This intermediate
does not exchange phosphate and is relatively stable at
pH values about 5.
It shows no net synthesis of urea
though its hydrolysis is more rapid in the presence of
ammonia, an inhibitor in the urease reaction (114).
completely hydrolyzed upon removal of the enzyme.
It is
Arsenate
acts similarly to phosphate but the usual decrease in
stability of the arsenate intermediate is noted.
The
character of this phosphate-containing intermediate is
presently obscure but the supposition of a compound like
CAP remains strong and some of the negative results ob­
tained in determining the existance of CAP may be due to the
techniques employed.
If these data do support a carbamyl transfer, the
search for an acceptor becomes a search for a substrate
for urease in defiance of the statement of Sumner and
Somersj "it acts upon urea and nothing else." (109).
It
may be possible that these workers have overlooked or did
not have available to them all ureido compounds.
Of course,
CAP has only recently been prepared (5*0 and it appears to
be hydrolyzed, though very slowly and only in the presence
of ammonia.
Of interest is that the enzyme prevents the
normal rate of nonenzymatic degradation of CAP to prevail
and that ammonia does not effect the nonenzymatic degrada­
tion.
Citrulline was used because it is involved in a
carbamyl transfer reaction.
Table XV indicates that citrul­
line is degraded and that carbon dioxide is released.
This
release is inhibited by phosphate and enhanced by arsenate
at pH 6.0.
Chromatography of the reaction mixture shows a
faint spot which has not been identified.
If it is ornithine
an oxygen-18 exchange experiment should be run.
Experi­
ments using ornithine as an acceptor are also important.
In further quest of an acceptor aspartate was examined.
Urea was degraded in the presence of aspartate and at least
66
one unique spot was found on the chromatogram.
This must
he re-examined to determine its relation to ureidosuccinic
acid.
The urease work presented here is incomplete in many
respects hut a new concept for its function can he seen.
Urease as a decarboxylase has heen reported (115).
The
idea of transfer by urease has heen hinted before (1 1 6 ,
1 1 7 ) and on the basis of these initial experiments, the
probability of a carbamyl transfering capacity remains as
an important consideration.
67
SUMMAKT
The mechanism of arsenate-activated enzymatic re­
actions have been studied*
The degradation of gluta­
mine in the presence of oxygen-18 labeled phosphate and
arsenate resulted in carboxyl labeled glutamate.
Arsen­
olysis of glycogen in the presence of oxygen-18 labeled
arsenate gave glucose with the carbon one oxygen labeled.
Carbon dioxide resulting from the citrulline breakdown in
the presence of oxygen-18 labeled arsenate and phosphate
contained oxygen-18.
In the arsenolysis of acetyl phos­
phate, oxygen-18 from arsenate labeled with oxygen-18
occurred in the acetate.
No oxygen-18 transfer occurred
in the arsenate enhancement of fumarase activity.
The nonenzymatic arsenolysis and phosphorolysis
were examined and the kinetics reported.
Acetyl arsenate
was identified and the preparation of other acyl arsenates
was mentioned.
The effect of arsenate and phosphate upon urease
activity was studied.
Several reactions were run with
the intention of determining a transfer function for urease.
Oxygen-18 labeled arsenate and phosphate were present in
the reaction in which urea was decomposed.
carbon dioxide contained oxygen-18.
68
The released
A discussion of the mechanism of arsenate-activated
reactions and arsenolysis reactions was presented 'which
included a proposal for a general mode of action.
The
effects of arsenate in vivo were correlated with the
enzymatic results.
A general theory on arsenate was
described.
69
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77
AUTOBIOGRAPHY
I, Donald Hillman Slocum, was b o m in Flushing, New
York-,on the sixth day of January, 1930.
I attended elemen­
tary school and high school in Flushing, New york,
Ify
undergraduate training was received at Davis and Elkins
College, Elkins,* West Virginia, which granted me the degree
j
Bachelor of Science in 1951.
I served in the United States
Army Corps of Engineers as a lieutenant.
from active duty in November, 1953.
Charles Pfizer and Company, Inc.
I was released
I was employed by
I attended the University
of Vermont and received a Master of Science degree in 1956.
While in residence there, I was a research assistant under
Dr. John E. Little, Department of Agricultural Biochemistry
My research program was supported by the Atomic Energy
Commission and by Charles Pfizer and Company, Inc.
In July
1956, I was appointed the Kettering Fellow at Ohio State
University, Department of Agricultural Biochemistry under
Dr. J. E. Varner.
I held this position for two years while
completing the requirements for the degree Doctor of
Philosophy.