THE EFFECT GE ALPHAcAMENCIBUTYNC
ACID AND PROPIONEC AC§D ON THE
SYNTHESE$ OF PYREMIDINES AND
ARGENENE ‘EN NEUROSPORA CRASSA

Thesis fu- er Denna of DEL, D.
MECEEGAN STATE UNEVEPZSETY
Donald Eugene Wampier
A965

THESIS

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ABSTRACT

THE EFFECT OF a-AMINOBCTYRIC ACID AND
PROPIONIC ACID ON THE SYNTHESIS OF PYRIMIDINES
AND ARGININE IN NEUROSPORA CRASSA

By Donald Eugene Wampler

For several years Dr. Fairley and coworkers have been studying
the growth of a pyrimidine-requiring mutant of Neurospora crassa.
This mutant, 1298, will grow on.a medium containing instead of
pyrimidines any one of a number of aliphatic acids including a—
aminobutyric acid and propionic acid. This thesis reports work
done in an attempt to understand how propionate and a-aminobutyrate
promote growth.

The first major finding was that the intracellular arginine
concentration fell from about 20 umoles per gram dry mycelia when
the mold was grown on uridine to as little as l umole per gram
when grown in the presence of a-aminobutyrate or propionate. Evi-
dence from other laboratories suggested that the biochemical defi~
ciency of this and related mutants is the formation of a pyrimldine-
specific supply of carbamyl phosphate. The reduction in arginine
caused by propionate or a-aminobutyrate apparently derepresses the
arginine-specific supply of carbamyl phosphate to the extent that
excess carbamyl phosphate can spill over into the pyrimidine
pathway.

On this basis, work was directed toward an explanation of how

the growth-promoting compounds reduce the concentration of arginine.

Donald Eugene Wampler

Besides the change in arginine concentration, a change in the
concentration of a number of other amino acids was found. These
changes are compatible with the idea that arginine production is
blocked in the formation of argininosuccinate. Crude extracts of
wild type E. crassa displayed about 2.5 units of argininosuccinate
synthetase activity per mg of protein. The enzyme was purified
approximately 15-fold by treatment with protamine, ammonium sulfate
fractionation, and chromatography on DEAE cellulose. Arginino-
succinate synthetase was strongly inhibited by inorganic pyrophos-
phate, L-arginine, AMP, ADP, and ATP. L-Valine, L—a-amino-n—
butyric acid, propionate, and L-threonine were also found to be
relatively effective inhibitors of the enzyme.

At least part of the reduction in intracellular arginine when
cells are grown on propionate or a-aminobutyrate can therefore be
explained as a result of the direct inhibition of argininosuccinate
synthetase by these growth-promoting compounds. These compounds
may also limit argininosuccinate synthetase activity indirectly by
increasing the concentration of valine, which also inhibits the
reaction. This explanation for the growth of 1298 on propionate and
a-aminobutyrate does not exclude the possibility that other metabolic
changes may contribute to the reduction in arginine concentration.

The inhibition of argininosuccinate synthetase by arginine
found in this work is the first demonstration of end product

inhibition in the pathway of arginine synthesis in Neurospora.

 

THE EFFECT OF a-AMINOBUTYRIC ACID AND
PROPIONIC ACID ON THE SYNTHESIS OF PYRIMIDINES

AND ARGININE IN NEUROSPORA CRASSA

BY

Donald Eugene Wampler

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1965

ACKNOWLEDGMENTS

I would like to thank Dr. James L. Fairley for his advice
and assistance during the work on this problem. He achieved a

happy balance of guidance and opportunity for independent
effort. I would also like to thank Dr. Rowland H. Davis for
several stimulating discussions and for permission to use some

unpublished data.

Finally, I would like to express appreciation to the
National Institutes of Health for financial assistance during

this project.

D.E.W.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . .

EXPERIMENTAL PROCEDURES. . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . .
Growth of Organisms . . . . . . . . . . . . . .
Preparation of Acetone Powders. . . . . . . . .
Assay for Free Intracellular Arginine . . .
Assays for Amino Acid Pool Sizes. . . . . . .
Preparation of Crude Extracts for Enzyme Assays

Assays for Urea Cycle Enzymes . . . . . . . . .

Partial Purification of Argininosuccinate Synthetase.

RESULTS. . . . . . . . . . . . . . . . . . . . .
Changes in Free Amino Acid Levels . . . . . . .
Intracellular Arginine Concentration. . . . . .
Enzymatic Studies . . . . . . . . . . . . . . .

General Findings . . . . . . . . . . .

Properties of Argininosuccinate Synthetase '

cmde Extracts . C O O O O O l C O C O O

Properties of the Purified Preparation .
DISCIJSSI ON 0 O O l O O O O O O O O O I O O O 0

REFERENCES 0 O O O O O O O O O O C 0

iii

19
19
23
26
26

28

33

38

45

LIST OF TABLES

TABLE . Page
I Differences Among the Pyr-3 Mutants . . . . . . . . . 1
II Effect of Growth Medium on Free Amino Acid Composi-
tion of g. crassa . . . . . . . . . . . . . . . . . . 21
III The Arginine Content of a Wild-Type Strain of EEEEQ“

spora crassa and the Pyrimidineless Mutant, Strain
1298, under Various Conditions. . . . . . . . . . . . 23

IV Activity of Urea Cycle Enzymes and Urease in Crude
Extracts of Neurospora. . . . . . . . . . . . . . . . 27

 

V Influence of Reaction Components on Argininosuccinate
Synthetase Activity from g. crassa. . . . . . . . . . 28

VI Inhibitors of Argininosuccinate Synthetase. . . . . . 32

iv

FIGURE

LIST OF FIGURES

Metabolic Pathways Involved in Pyrimidine and
Arginine Synthesis in Neurospora crassa . . . . .

Protein and Argininosuccinate Synthetase Activity

_ Recovered from DEAE Cellulose Column. . . . . . .

Effect of a-Aminobutyrate on the Free Amino Acid
Composition of N. crassa 1298 . . . . . . .

The Relationship Between the Concentration of
a-Aminobutyrate and of Propionate in the Culture
Medium and the Arginine Content of the Mycelium .

Factors Affecting Argininosuccinate Synthetase
Measured in the Crude Preparation . . . . . . . .

Factors Affecting Argininosuccinate Synthetase
Measured in the Crude Preparation . . . . . . .

Raw Data from Spectrophotometric Assay. . . . . .

Factors Affecting Argininosuccinate Synthetase
Measured in the Partially Purified Preparation. .

Page

17

20

25

30

31

35

36

INTRODUCTION

Among the more than 380 Neurospora mutants isolated by Beadle and

 

Tatum (1), some #5 required pyrimidines for growth. On the basis 0f.
complementation data, Houlahan and Mitchell (2) arranged a number of
these mutants into 3 linkage groups: pyr-l, pyr-2, and pyr-3. All
pyr—3 mutants require pyrimidines when grown at 35°, but when grown at
25°, they fall into three phenotypic classes (3): a, b, and c. At
25°, pyr-3a requires the normal supplement of pyrimidines, pyr-3b

grows on basal medium, and pyr-3c requires about 1/6 the normal pyri-

midine supplement (see Table I).

 

 

Table I. Differences Among the Pyr-3 Mutants
Growth on Suppress- RNA*(2)

”Uta“ Numb" ozABA (u) ibility (3,9) ATC (9'11) Requirement

3a 37301 + + + 3.3

1298 + + +

3b 37815 + + + 0

3c 67602 + 0.38

3d 45502 and 0 0 0 3.3

several others

 

* mg hydrolyzed ribonucleic acid required for one-half wild growth
at 25°.

Abbreviations: ATC - Aspartic transcarbamylase
aABA .- a-Aminobutyric acid.
While Houlahan and Mitchell were working on the characteristics of
these mutants, one strain_of pyr-3a began to grow as if a back mutation
had occurred (2). A_cross between this apparent reversion and the wild

type produced some asci which contained the expected distribution of

mutant and wild spores but also another type of ascus which could not

be explained on the basis of a back mutation. This unexpected ascus
could be explained, however, if a second, independent mutation had
occurred which suppressed the pyr-3a mutation. Further study showed

that a second mutation had, indeed occurred. The presence of this
suppressor mutation, s, renders the otherwise pyrimidine-deficient
mutants phenotypically.wi1d-type and, therefore, the gene can be detected
only in conjunction with the mutation which it suppresses. The suppressor
phenomenon led to the identification of a fourth member of this allelic
group, pyr-3d. Pyr-3d has the same nutritional requirements as pyr-3a
but is not suppressible.

There are also two nutritional situations which, like the suppressor
mutation, allow pyr-3a mutants to grow in the absence of pyrimidines.
Fairley £3 31. (4,5) have shown that 1298 will grow on basal medium
supplemented with any one of seVeral aliphatic acids. These compounds,
including a-aminobutyrate, propionate, and threonine, will also
support growth of another pyr-3a mutant, 37301, and the pyr-3b mutant,
but not the pyr-l or pyr-2 mutants. Recently, Charles (6,7) has found
that strain 1298 can be made to grow on basal medium by increasing the
carbon dioxide concentration.

Davis made the first attempts to explain the pyrimidine mutation
in terms of enzymatic activity. He found (8) that pyr-l and pyr-2 are
blocked between carbamyl aspartate and uridylic acid (reactions 3, a,

S, and 6 in Figure 1). Even though the pyr-3 mutants will not grow
when carbamyl aspartate is added to the medium, they were shown to

contain all the enzymes required for the conversion of carbamyl aspartate

' 3

Figure 1. Metabolic Pathways Involved in Pyrimidine and Arginine
Synthesis in Neurospora crassa

 
 
  

 

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Footnotes for Figure l:

 

 

Reac-
tion E. C. No. Mutant Ref. Comments
1 2.7.2.2 pyr-3a*,arg-3 12,41 2 enzymes in N. crassa
2 2.1.3.2 pyr—3d 9,43 same as 2.7.2.2 in N. crassa
3 3.5.2.3 pyr—l* 8
4 1.3.3.1
5 2.4.2.10 pyr-2 8
6 pyr-4 8
7 2.1.3 3 arg—12 l3
8 6.3.4.5 arg-l 25
9 4.3.2.1 arg-lO 25
10 3.5.3.1
11 3.5.1.5
12 1.4.1 3-4 prol-2 or 3*
13 2.6.1.1-8 prol-3 or 2*
14
15 2.6.1.13
16 arg-4
17
18 erg-6*
19 12 nonenzymatic
20 prol-l
21 2.7.2.4 44 3 enzymes in E. coli.
22
23 1.1.1.3
24 4.2.1.15
25 2.7.1.39
26 4.2.99.2
27 4.2.1.16 27 inhibited by isoleucine,
released by a-aminobutyrate
28
29
30
31 group 4 28,45 stimulated by growth on a-keto-
butyrate,threonine,and isoleucine.
32 group I 45
33 group 2 and 3* 45
34

 

 

*The enzymes affected by these mutations have
not been established.

to pyrimidine nucleotides. Davis, therefore, assumed that the pyr-3
mutants are blocked in or before carbamyl aspartate formation. Pyr-3
mutants are not blocked in aspartic acid formation since they do not
require this amino acid. Neither do they seem to be deficient in
carbamyl phosphate formation since they do not require arginine. The
only enzyme left, then, is aspartic transcarbamylase.

In 1960, Davis showed that the pyr-3d mutants lack aspartic
transcarbamylase activity (9) while the pyr-3a and pyr-3b mutants have
normal activity. The enzymatic data complement the suppressor data
exactly -- those mutants which are suppressible have aspartic trans-
carbamylase; those mutants which are not suppressible do not have
aspartic transcarbamylase. So, the pyr-3a mutants presented a dilemma.
Although they seemed to have all of the enzymes necessary for pyrimi-
dine synthesis, they nevertheless required pyrimidines.

Fairley and Adams and Davis simultaneously reported two related
observations which provided one of the first clues to solving this
dilemma. Fairley and Adams reported (10) that growth of 1298 on a-
aminobutyrate, like growth of the suppressed mutant, was strongly
inhibited by arginine. Davis reported (11) that the suppressor gene
caused a reduction in the intracellular concentration of arginine. In
both cases arginine antagonized growth. Since normal cellular con-
centrations of arginine apparently inhibited pyrimidine formation,
Davis proposed that some step in pyrimidine synthesis was abnormally
sensitive to arginine.

Shortly thereafter, Davis discovered (12) that suppressed mutants

exhibit low ornithine transcarbamylase activity. This production of

an altered, less active form of ornithine transcarbamylase finally
allowed the suppressor to be identified independent of the pyrimidine
requirement. Further work by Davis and Thwaites (l3) established that
s is the structural locus for ornithine transcarbamylase.

Work with the suppressor gene, and other data to be discussed later,
led to an explanation of the pyr—3a mutation originally put forth by
Davis (12,14), and later by Charles (7). This explanation postulates
two sources of active carbamyl groups in N. crassa; one specific for
arginine production and the other for pyrimidine production. The pyr-3a
mutants presumably lack the pyrimidine-specific enzyme. Conditions
such as the suppressor mutation which limit arginine production de-
repress the arginine-specific enzyme to the extent that the extra
carbamyl phosphate can be used in pyrimidine synthesis.

The problem of pyrimidine synthesis in pyr-3a mutants thus became
a problem in the control of arginine synthesis. This thesis reports
studies on the effect of propionate and aminobutyrate on the synthesis

of arginine and pyrimidines.

EXPERIMENTAL PROCEDURES

Materials

Neurospora crassa strains used in this work were the wild type,

 

1A, and a pyr-3a mutant, 1298. These strains are among those in the
collection at Dartmouth College. All reagents were obtained from
commercial sources.
Growth of Organisms

Organisms were grown in liquid shake culture in 2500-ml Fernbach
flasks on a Eberbach reciprocal shaker at 86 strokes per minute.
Mycelia used in studies of arginine concentration were grown at room
temperature in flasks containing 500 ml of modified Fries medium (15).
Mycelia used for enzymatic studies were grown for 22 hours from
conidial inocula at 32 - 35° in flasks containing 750 ml Vogel's N
medium (16) and 15 g sucrose. In addition, supplemented medium con-
tained 80 mg uracil or 200 mg D,L-a-aminobutyric acid or 200 mg sodium
propionate per liter, unless otherwise indicated.
Preparation of Acetone Powders

Mycelia were harvested by filtering with suction over 4 layers of
cheesecloth and washing several times with distilled water. The moist
pads were torn into chunks and homogenized for about 30 sec. in a
Servall Omnimixer containing acetone which had been dried over Na2S04.
For free arginine assays and preliminary enzymatic studies, the acetone
was at room temperature. For later studies on argininosuccinate
synthetase, the acetone was kept at -20° in a dry ice-acetone bath.

The homogenized mixture was filtered with suction over Whatman No. l

filter paper and washed several times with dry, room-temperature acetone.

In those cases when the mycelial cake was not white at this stage
or when the initial homogenization was performed at -20°, the cakes
were again homogenized in dry, room temperature acetone, filtered,
and washed several times with dry acetone. The final white cake was
broken up and allowed to air dry at room temperature. The resulting
powder was white and very powdery. It could be stored at room tempera-
ture for amonth or so without change in arginine concentration.
Acetone powders used in enzyme studies were stored at 2 - 5° and were
always used within a week after harvesting with no noticeable change
in enzymatic properties.
Assay for Free Intracellular Agginine

With the use of a Tenbroeck glass tissue grinder, 50 mg of acetone
powder was extracted into 57ml of cold 10 percent trichloracetic acid
(TCA). The mixture was centrifuged in a clinical centrifuge and the
supernate saved. The residue was resuspended in 3 ml of TCA which
had been used to rinse out the tissue grinder and the mixture was again
centrifuged. The combined supernates were centrifuged for 10 min. at
4,400 times gravity. This final supernate was poured through a small
wad of glass wool and followed by 2 m1 of TCA, The resulting Solution
was adjusted to pH 8.0 (as determined with pH paper) with SN NaOH and
diluted to a volume of 15 ml. Aliquots of this solution were assayed
by a modified Sakaguchi procedure (17).
Assays for Amino Acid Pool Sizes

The amino acid concentrations of mycelial extracts were determined

by two methods: two—dimensional paper chromatography and analysis on

10

a Beckman Model 1208 amino acid analyzer.

For paper chromatograms, amino acids were extracted from 300 mg
of acetone powder with 10 percent TCA as described above. Instead of
neutralizing with NaOH, the excess TCA was removed by extracting
several times with ether and the solution was then concentrated to about
1 ml with a rotary evaporator at a temperature not greater than 50°.
The concentrated solution was filtered and the flask washed with 3
small volumes of 10 percent isopropanol in water.

Two dimensional descending chromatograms were run in a chromato-
graphy cabinet. For the first direction, the solvent was a 12:5:3
(v:v:v) mixture of butanol, acetic acid, and water. The second direc-
tion was run with a 4:1 (v:v) phenol-water mixture with a petri dish
containing 2N NH4OH in the bottom of the cabinet.

Samples assayed on the amino acid analyzer were extracted from
growing mycelia rather than acetone powders. A 350 ml portion of the
shake culture (corresponding to about 800 mg of acetone powder) was
filtered through Whatman No. 1 filter paper and the mycelial pad washed
with several portions of water. The moist pad was homogenized for 20
seconds in 25 ml of cold 1 percent picric acid using a Servall Omni-
mixer. This suspension was further homogenized in a glass Tenbroeck
tissue grinder until the mycelial fragments passed the walls freely
and then was centrifuged for 2 min. in a clinical centrifuge.

Picric acid was removed on,a 2.2 x*3 cm column of Dowex 1eX12,
100 - 200 mesh, in the chloride form. Amino acids were washed off of

the column with 0.02 M HCl and the combined effluents were reduced to

11

a small volume (about 1 ml) on a rotary evaporator. The concentrated
sample was filtered and the flask washed with small samples of 0.02 M
HCl. The final volume of the extract was about 5 ml.
Preparation of Crude Extracts for Enzyme Assays

Extracts used in the initial survey of urea cycle enzymes were
prepared by grinding 200 mg of acetone powder in 10 m1 of 0.02 M tris-
acetate buffer, pH 8.0, with the aid of a Tenbroeck glass homogenizer.
The solids were collected by centrifugation and were resuspended in
6 m1 of the same buffer. The combined extracts were diluted to two mg
protein per ml.

Extracts used in later studies of argininosuccinate synthetase
activity were prepared by homogenizing 2 g acetone powder in 18 ml
0.02 M tris-acetate buffer, pH 8.0, for one minute using a Servall
Omnimixer. The thick paste was centrifuged and the residue resuspended
in 5 ml of the same buffer. The combined extracts were run into a
2.2 x 25 cm column of Sephadex G-25, coarse grade, and eluted with
0.01 M phosphate buffer, pH 7.4. Fractions containing more than 2 mg
protein per ml, as measured by the biuret reaction (18), were combined
and diluted to 2 mg protein per m1.
Assays for Urea Cycle Enzymes

In all expressions of activity, one unit was defined as the amount
of enzyme catalyzing the production of 1 umole of product (or removal
of one umole of substrate) per hour under the specified conditions. All
protein measurements were made by the biuret method (18). Citrulline
and urea were measured by the method of Archibald (19), as modified by

Gerhard and Pardee (20).

12

An attempt was made to measure H01403‘ incorporation into
citrulline but without success. Since a successful assay for the
carbamyl phosphokinase reaction has now been reported (21), the
unsuccessful methods studied in this laboratory will not be described
here.

Ornithine transcarbamylase activity was measured essentially as
described by Davis (22). The incubation mixture contained in a volume
of 2.6 ml: 20 umoles L-ornithine: 20 umoles carbamyl phosphate; 500
umoles tris-acetate buffer, pH 9.0; and 0.04 ml of mycelial extract.
'After incubation of the mixture for 15 minutes at 28°C, the reaction
was stopped with 0.5 ml of 2N perchloric acid. The precipitate was
removed by centrifugation and 1.0 m1 portions of the supernatant
solutions were analyzed for citrulline.

Argininosuccinase was detected by the formation of arginino-
succinate from arginine and fumaric acid (23). The reaction mixture
contained in a volume of 1.6 ml: 80 umoles potassium fumarate: 80
umoles L-arginine; 100 umoles phosphate buffer, pH 7.0: and 0.4 ml of
the enzyme extract. After incubation for an hour at 28°, the reaction
was stopped by boiling for 5 minutes. Coagulated protein was removed
by centrifugation, and 25 ml of the supernate was spotted on Whatnan
No. 4 filter paper. Argininoaiccinate was separated from other constitu-
ents by ascending chromatography. The liquid phase was water-saturated
phenol containing 2 drops of 4N NH4OH per 25 ml. After the chromatograms
were dried, ninhydrin spray was used to locate the amino acids. No
quantitative measurements were made other than visual estimation of

the size of the spot corresponding to argifinxmccinate.

l3

Arginase was measured essentially as described by D. M. Greenberg
(24). The reaction mixture contained in a volume of 2 ml: 50 umoles
arginine: 400 umoles tris-acetate buffer, pH 7.4; and 1 ml of the
enzyme extract which had been preincubated with manganese for 1/2 hr
at 40°. The reaction was allowed to proceed for 20 min. at 28° and
then stopped by adding 0.5 m1 2N perchloric acid. Denatured protein
was removed by centrifugation. Arginine was removed by passing a
1 ml sample of the supernate through a 1.5 x 1.5 cm column of Dowex
50-W, 100-200 mesh, hydrogen form. The column was washed with a 2 ml
and then a 1 m1 portion of water. One ml of the eluate from the
column was assayed for urea.

Urease was measured by the disappearance of urea. The reaction
mixture contained in a volume of 2 ml: 0.5 umoles urea; 100 umoles
phosphate buffer: and 1 ml of the enzyme extract. The mixture was
incubated for 30 min. at 28° and the reaction was stopped by the
addition of 1 m1 of 1N perchloric acid. Denatured protein was
removed by centrifugation and 1 ml of the supernate was assayed for
urea.

Argininosuccinate synthetase activity was measured in two ways.
In what will be called the colorimetric method, activity was followed
by measuring citrulline disappearance (25). This assay was always
used to measure activity in crude extracts and occasionally was used
to measure activity in the purified preparation. In what will be called
the Spectrophotometric method, activity was measured through a series

of reactions to the oxidation of NADH*(26). The AMP formed by arginino-

 

fAbbreviations used in the text are: AMP, ADP, and ATP, adenosine 5'—
mono, di, and triphosphate, respectively; DEAE, diethylaminoethyl;
NADH, reduced nicotine adenine dinucleotide; tris, tris(hydroxymethyl)-
aminomethane.

14

succinate synthetase was converted to ADP by myokinase; the ADP then
accepted the phosphate of phosphoenol pyruvate to give pyruvate and the
latter was reduced by NADH to give lactate. The oxidation of NADH was
followed by the decrease in absorbance at 340 mu. Since inorganic
pyrophosphate strongly inhibits arghflreuccinate synthetase, inorganic
pyrophosphatase was also added to the spectrophotometric assay.

For the colorimetric assay, the reaction mixture contained in a
volume of 2 ml: 40 umoles potassium L-aspartate, pH 8.0; 20 umoles
MgSO4: 1.5 umoles L-citrulline; 2.0 umoles ATP; 20 umoles 3-phosphog1y-
ceric acid; 100 umoles tris-acetate buffer, pH 8.2. Enough crude extract
to contain 1 mg protein gave a convenient activity. The mixture was
incubated at 37° for 10 minutes and the reaction stopped with 1 m1 of
1N H0104. Three m1 of water was added (total volume 6 ml), and the mix-
ture was centrifuged to remove the denatured protein. A 1 ml sample
of the supernate was then assayed for citrulline.

For the spectrophotometric assay, the reaction mixture contained
in a volume of 1 ml: 0.50 umoles ATP; 2.08 umoles phosphoenolpyruvate;
0.47 umoles NADH; 10 umoles potassium L-aspartate; 10 umoles L-citrulline;
2.0 umoles MgSO4; 100 umoles tris-acetate buffer, pH 8.2; 150 units
myokinase; 125 units inorganic pyrophosphatase; 150 units lactic
dehydrogenase containing pyruvate kinase; and 3 - 5 units of arginino-
succinate synthetase.

Any assay which measures substrate disappearance has the disad-
vantage that the substrate concentration must be kept low so that the

amount consumed in the reaction will be a significant fraction of the

15

total. The citrulline concentration in this assay was about 10 percent
of the optimal concentration. Aside from this problem, the assay for
citrulline requires an hour and a half to run and the reaction cannot
be followed continuously.

The spectrophotometric assay has the disadvantage that there are
five enzymes involved in the assay. Argininosuccinate synthetase was
kept limiting by adding relatively large concentrations of the other
four enzymes. An indirect check of their activities was obtained by
removing the test cell and following the rate of reaction in the
control cell. Because there is a very rapid background reaction in
crude extracts, the spectrophotometric assay can only be used on
partially purified extracts.

NADH is relatively rapidly removed from the reaction mixture even
without the addition of aspartate, citrulline, or ATP. This backgfiound
reaction was measured in a control cell which contained all of the reagents
except citrulline. The removal of NADH in the test cell, which contained
all of the reagents, was due to the background reaction plus the series
of reactions starting with argininosuccinate synthetase. The difference
in the rate of NADH oxidation in these two cells was measured directly
on a Beckman DB spectrophotometer. This difference rate is twice the
rate of argininosuccinate synthesis since 2 moles of ADP are produced
with each mole of argininosuccinate synthesized.

Partial Purification of Argininosuccinate Synthetase
All solutions were kept between 0° and 5°. Using a Servall Omni-

mixer, 20 gm of acetone powder was homogenized for 30 sec. in 120 ml

16

of 0.02 M phosphate buffer, pH 7.4. The thick paste was allowed to
soak for 5 min. and then centrifuged 15 min. at 23,000 times gravity.
The supernate was saved and the residue redxtracted in 50 m1 of the
same buffer and centrifuged for 10 min. The supernates were combined
and the protein concentration adjusted to 10 mg per ml.

The solution was adjusted to pH 6.5 and made 0.1 M in ammonium
sulfate (0.03 saturation). One-tenth volume of a freshly prepared
4 percent protamine sulfate suspension was added dropwise with constant
stirring and the mixture stirred for an additional 5 min. The preci-
pitate was removed by centrifuging for 10 min. at 23,000 times gravity.

The supernate was brought to 0.36 saturation by slowly adding
solid ammonium sulfate (200 mg/ml). The mixture was stirred for an
additional 15 min. and the precipitate removed by centrifuging for 10
min. This supernate was made 0.71 saturated by adding more solid
ammonium sulfate (240 mg/ml), stirred for 15 min. and centrifuged.

The protein precipitated between 0.36 and 0.71 saturation was
dissolved in a small volume of 0.01 M phosphate buffer, pH 7.0. This
resuspended sample was dialyzed for several hours against four changes
of 0.01 M buffer and then dialyzed overnight.

The dialyzed extract was applied to a 2.2 x 15 cm column of DEAE
cellulose. Elution, using phosphate buffer, pH 7.0, was carried out
by the addition of 15 ml of 0.01 M buffer, 50 ml 0.02 M buffer, and then
a linear gradient ranging from 0.02 M to 0.06 M. The elution rate was
held at 2 ml per minute (see Figure 2).

A small increase in activity could be achieved by putting this

17

Slope of Reaction Using 0.2 ml of Fraction

o.m

masHo> acosaumm H8

 

 

 

 

 

 

ow: 00: can cam 00H ow
q d 1 w h A t .+T
. .:.o
r lwoo
r nN.H
fi 1©.H
fl io.N
v 10.:
mommnm0m2< in
dmmuonuczm oumcHoosmochkum It. no.0

Houucoo it

casaoo moodsaaoo m<ma Bonn
oouo>ooom muw>fluo< ommuocucmm oumcwoosmocwawwm< ecu awmuomm .N ouswwm

nm ogz is aousqaosqv

18

preparation through a second DEAE cellulose step, using a 1.1 x 10 cm

column. Since the only purpose of purification was to get a prepara-

tion which could be conveniently measured in the spectrophotometric

assay, this second DEAE step was not normally used.

RESULTS

Changes in Free Amino Acid Levels

Growth of both the wild type organism and the mutant 1298
on o-aminobutyrate or propionate caused changes in the intra-
cellular concentrations of several amino acids. Changes in the
amino acid composition introduced by growth in the presence of
ofaminobutyrate are illustrated in Figure 3. Changes in neutral
and acidic amino acids when mycelia were grown with propionate
are listed in Table II. The automatic amino acid analyzer gave
good separation for most amino acids. The time (measured as
elution volume) at which each amino acid came off of the column
was the same from one extract to another but extracted amino
acids came off progressively slower than amino acids from a
calibration mixture. Identification was further complicated by
the presence of several unidentified peaks and shoulders.

The amino acid composition of an extract from mycelia grown
in the presence of o-aminobutyrate is also presented in Table II.
Although there was a substantial increase in isoleucine and B-
alanine concentration, this extract shows no increase in valine
and a relatively small increase in the peak which should contain
o-aminobutyrate. It is likely that this culture was allowed to
grow too long and the organism had already removed most of the
o-mminobutyrate.

Both the paper chromatograms and the amino acid analyzer
show a large increase in the concentration of isoleucine when

the mold is grown with o-aminobutyrate. The paper chromatograms

19

20

 

 

Effect of a-Aminobutyrate on the Free Amino Acid

Uridine and o-Aminobutyrate

 

 

Q

@
O®@

8

0

Figure 3.
Composition of N. crassa 1298
The Growth Medium is Supplemented with:
Uridine
o
@A
O
“
m
‘3 o
3 0~
'L ‘D
8
,2 Q9
“@ .0
0 c::D
09

 

 

 

A
r

Butanol-acetic acid-water

 

 

0, 2390.,

//

Letters refer to the position of known amino acids run on a

similar chromatogram.

Abbreviations are: A, aspartate;

Al, alanine: Ar, arginine: o, a-aminobutyrate; C, citrulline:
G, glutamate; I, isoleucine; 0, ornithine; V, valine.

21

 

 

 

Table II. Effect of Growth Medium on Free Amino Acid Composition
of N. crassa

. . Concentration1 of Amino Acid when

Amino Acid2 3:3;2222 Mycelia were Grown on:
Basal- oABA Propionate4

Unknown 55 -3.5 8.9 .9.2
Aspartate 117 32.4 48.8 24.5
Serine 153 ----- Off Scale --------
Unknown 171 7.0 3.9 13.8
Glutamate 182 ----- Off Scale --------
'Glycine 247 31.1 29.0 29.8
Alanine 274 ----- Off Scale --------
Unknown5 306 17.0 22.2 4.8
Valine 311 18.4 17.0 33.4
Unknown 316 41 . 7 no. 7 13.u ‘
Methionine 332 1.4 3.8 1.4
Isoleucine 348 4.1 40.5 6.3
B-alanine 518 1.0 9.7 1.5

 

Concentration was calculated by multiplying the width at one-

half the height by the height of the peak. Concentration units
are different for each amino acid so comparisons can be made
between extracts but not between amino acids.

Leucine, tyrosine, and phenylalanine could also be identified

but they were present in small amounts and there was little
difference in concentration among the three extracts. There
were three peaks which were off the scale. The largest peak

was alanine.

3 Position is measured in ml of effluent from the column.

4 The extract from mycelia grown with sodium propionate was less
concentrated than the other two extracts. Values in this column
are the raw data multiplied by 2.5, a factor which brings the
glycine value to that of the other extracts.

5 May contain o-aminobutyrate.

~‘—-—~

22

also show an increase in valine and obaminobutyrate. The reduction
in arginine concentration, shown by the paper chromatograms, was
studied in detail and will be discussed later. The amino acid
analyzer shows an increase in valine and a slight increase in iso-
leucine when mycelia were grown with propionate. There is, however,
no increase in the peak which may contain a-aminobutyrate.

Hayashibi and Uemura (27) have demonstrated that isoleucine

exerts feedback control on its own synthesis in Bacillus subtilis

 

by inhibiting threonine deaminase (reaction 27). They further showed
that o-aminobutyrate releases this inhibition and cells grown in
the presence of o-aminobutyrate accumulate "a large quantity of
L-isoleucine." The large increase in isoleucine concentration in
cells grown on o-aminobutyrate suggests that a similar situation
exists in N. crassa.

Horvath gt 31. (28) have shown that the presence of threonine,

o-ketobutyrate or isoleucine in the growth medium of Pseudomonas

 

aeruginosa or Escherichia coli increases the a-acetolactate forming
system (reaction 31) which is the first reaction peculiar to valine
and isoleucine synthesis. Since o-ketobutyrate and threonine can
.readily be formed from o-aminobutyrate (reactions 30 and 27), and
isoleucine synthesis is increased with growth on o-aminobutyrate,
the logical conclusion is that a similar explanation for the valine
increase applies with N. crassa. It is more difficult to explain
the increase in valine found when the mold was grown on propionate.

Attention must be drawn to the fact that the actual inducer in

23

the experiments of Horvath gt 21. is unknown. Conceivably, this
could be propionate formed by decarboxylation of o-ketobutyrate.
Intracellular Arginine Concentration

The arginine concentrations in mycelial powders of strains
1A and 1298 grown under various culture conditions appear in
Table III. Because the molds grow at considerably different rates
under different culture conditions, it was necessary to assay for
arginine at comparable stages of growth. Gaps which appear in the
data of the Table result chiefly from difficulties in harvesting
the mold at the desired stages.
Table III. The Arginine Content of a Wild-Type Strain of Negro-

sppra crassa and the Pyrimidineless Mutant, Strain
.1298, under Various Conditions

 

 

Arginine Content1 at

 

Supplements to 500 ml. Strain of Various Growth Stages2
of Fries medium the Mold
0.1—00u9 005-0099 100-205
None Wild 20 -- 20
40 mg. Uridine Wild 33 23 20
1298 28 31 30
100 mg. Aminobutyric Wild 10 18 20
acid 1298 4.5 -- 24
40 mg. Uridine + 100 Wild 10 12 16
mg. Aminobutyric 1298 -- 7.5 22
acid

100 mg. Sodium Pro- Wild 1.2 5.0 18
pionate 1298 0.9 -- 4
100 mg. Sodium Pro- Wild -- 6.3 --
pionate + 40 mg. 1298 1.9 l 2 --

Uridine

 

1 The content of arginine is expressed as umoles per gram of
mycelial acetone powder.

3 The stage of growth is expressed as grams of mycelium obtained
after harvest and extraction with acetone.

24

The level of free arginine in wild-type mycelia grown on basal
medium was about 20 umoles per gram of acetone powder. This level
was not affected appreciably by the stage of growth or the presence
of uridine, except for an apparent increase in the arginine concen-
tration at early stages of growth with uridine. In all cases, when
uridine was the supplement, the arginine content of the mutant strain
was about 30 umoles per gram.

For both strains of the mold, the presence of either propionate
or aminobutyrate resulted in low arginine concentrations, regardless
of the presence or absence of uridine. Levels as low as 1 umole
per gram of acetone powder, 5 percent or less of normal, were
obtained with propionate. These effects were most pronounced in
early growth, presumably because obaminobutyrate and propionate
are removed by metabolic processes as growth proceeds.

Figure 4 summarizes data which were obtained from experiments
designed to determine the relationship between the o-aminobutyrate
or propionate concentration in the culture medium and the intra-
mycelial concentration of free arginine. In these experiments,
sufficient uridine (80 mg. per liter) was present to provide maxi-
mal growth rates regardless of the concentration of propionate or
o-aminobutyrate. All mycelia were harvested at essentially the
same early stage of growth.

It may be seen that for the concentration range examined, the
arginine content proved to be dependent in inverse fashion upon

the logarithm of the concentration of either propionate or o-amino-

25

Figure 4. The Relationship Between the Concentration of o-Amino-
butyrate and of Propionate in the Culture Medium and
the Arginine Content of the Mycelium.

 

 

 

 

24[
a i
20 _ ' I
. o

g 16 _
o
4.1
C
o
O 12_
o
C
"-4
.5 8
w .
u
‘<

.4_

0 n

0.8
Logarithm of
Supplement Concentration
o propionate o a-aminobutyrate

Arginine content is given in umoles per gram of
mycelial acetone powder. The supplement concen-
tration is given in millimoles per liter.

26

butyrate. In these experiments, the o-aminobutyrate used

was the DL mixture. It is known that only the L-isomer is capable
of supporting growth of the mold (5). If the data for o-aminobuty-
rate given in Figure 4 were replotted under the assumption that
only the L-isomer is effective in lowering the arginine concen-
tration, the line for o-aminobutyrate would approach closely that
for propionate. This suggests, rather surprisingly, that the two
substances may be equally potent on a molar basis in reducing

the arginine content of the mycelium.

Enzymatic Studies

 

General Findings. All of the enzymes involved in the Krebs
urea cycle (reactions 7-10) can be demonstrated in extractsiof
1:1. crassa (Table IV). During the early stages of this work when
the data in Table IV were gathered, crude extracts exhibited only
about 0.2 units of activity per mg protein. Later, after making
several changes in the preparation of extracts and-assay condi-
tions, crude extracts consistently exhibited 2.2 to 2.6 units per
mg protein.

The optimal conditions for measuring ornithine transcarbamy-
lase activity had been worked out previously (31). Although
adding large amounts of arginine to the growth medium does not
repress ornithine transcarbamylase activity (29), conditions
which reduce the intracellular arginine concentration do cause
derepression, as shown in Table IV.

The last three enzymes listed in Table IV were not studied

in detail.

27

Table IV. Activity of Urea Cycle Enzymes and Urease in Crude
Extracts of Neurospora

 

 

 

Enzyme No'1 This Thesis DIE::VW:¥: Derepressed3_
CPK l 0 0.2 (29) 0.3 Arg 3(29)
OTC 7 20 20 (12) 85 Arg 3(12)

68 o-ABA

65 Prop.
ASSase 8 0.2 to 2.6 0.6 (25)
ASAase. 9 + 0.3-0.8 (25)
Arginase 10 3.0 7 (30) 25 Arginine(30)
Urease ll , 0.8

1 Reaction numbers in Figure 1.
3 Activity is umoles of product formed per mg protein per hour.

3 These activities are either derepressed or induced. The N.
crassa mutant or the supplement used to cause this change is
also given.

NOTE: Numbers shown in parentheses refer to references.

Abbreviations used in Table IV:

CPK - Carbamyl Phosphokinase
OTC - Ornithine Transcarbamylase
ASSase - Argininosuccinate Synthetase

ASAase - Argininosuccinase
a-ABA - o-Amino-n-butyric Acid

Prop. - Sodium Propionate

28

Properties of Argininosuccinate Synthetase in Crude Extracts.
The argininosuccinate synthetase activity found in crude extracts
was absolutely dependent upon the presence of aspartate, citrulline,
ATP, and magnesium (Table V). Addition of inorganic pyrophosphatase

Table V. Influence of Reaction Components on Argininosuccinate
Synthetase Activity from N. crassa

 

Percent Control Activity

 

 

 

 

 

Components of the Colorimetric Spectrophotometric
Reaction Mixture Assay Assay
ASSase Back.
Standard 100 100 100
Standard less aspartate 0 0 100
Standard less citrulline * 0 100
Standard less ATP 0 23 104
Standard less magnesium 0 0 0
Standard less enzyme 0 0 0
Standard less 3-PGA 60 *
Standard plus PPase 104 * *
Standard with double 3-PGA 100 * *
Standard less PPase * 85 100
Standard less PEP * 0 0
Standard less myokinase * 0 100
Standard less LDH * 0 0
Stangsig,mggfiegizincubated * 87 85
Abbreviations used in Table V: *Does not apply.
ASSase - Argininosuccinate Synthetase
Back. - Reaction in Control Cell
3-PGA - 3-Phosphog1yceric Acid
PPase - Inorganic Pyrophosphatase
PEP - Phosphoenolpyruvate

LDH - Lactic Dehydrogenase

29

increased the activity slightly, but this enzyme was not normally
added. Doubling the concentration of 3-phosphog1yceric acid did
not increase the activity. The activity was linear with respect
to time for at least 10 minutes and with respect to enzyme concen-
tration within the range used (Figure 5a). The pH-optimum was
measured using 3 buffers (Figure 5b). There is little difference
in activity between pH 7.4 and 8.2.

Figure 6a shows the relationship between activity and ATP
concentration. Adenosine triphosphate is both a substrate and
inhibitor of argininosuccinate synthetase. Concentrations either
above or below 10'3 M cause a decrease in activity. Other
inhibitors are listed in Table VI.

Of the compounds which allow growth of the pyr-3a mutants --
propionate, o-aminobutyrate, and threonine -- only o-aminobutyrate
can be called a good inhibitor (see Figure 6b also). Two products
of the reaction -- pyrophosphate and AMP -- also inhibit. Inor-
ganic pyrophosphate is a weak inhibitor when it is added to the
reaction with the substrates (upper curve, Figure 6b), but it is
a strong inhibitor if it is preincubated with the enzyme for 10
minutes before the substrates are added (lower curve, Figure 6b).

Valine and o-aminobutyrate were the only amino acids which
inhibited more than 50 percent. The fact that valine inhibits

argininosuccinate synthetase probably explains the observation

30

Figure 5. Factors Affecting Argininosuccinate Synthetase
Measured in the Crude Preparation

 

 

 

 

 

5b. pH
5a. Time and Enzyme 3
Concentration T
160
r
>.
4..)
"-4
.3
45 120- 2,.
<
c (D
:3 80. u
'o -H
c c
m D
(7) no 0
u _ 1.
C:
o
8
0 0 1 1 l 1
m 0 0.5 1.0 1.5 2.0
I 5 10 15 20
0 I l l 4
0 Enzyme concentration in 7 .8 9 10

mg protein per ml.

I Time in minutes. 0 Phosphate buffer.

x Standard conditions. ,x glyc1ne-NaOH buffer.

a tris-chloride buffer.

For figure 5b, units are umoles citrulline removed under standard
conditions (see Methods).

31

Figure 6. Factors Affecting Argininosuccinate Synthetase
Measured in the Crude Preparation

6a. ATP 6b. Inhibitors

100 - 100 — ‘\\\\\\\\d

15‘
XI

     

80 e 80

 

 

 

>1
H
-.-4
>
n4
4.:
(J
‘< 60 ~ 60
'0
N
a
'U
S 40 ~ no
4.:
U2
4.:
8 20 20
U
H
0
p" o l l l O
l 2 3

ATP M x 103 Inhibitors M x 103

6b. gf inorganic pyrophosphate, o inorganic pyrophosphate
preincubated with enzyme for 10 minutes, 0 L-o-amino-

butyrate, .6 L-valine.

32

Table VI. Inhibitors of Argininosuccinate Synthetase

 

 

 

 

 

Inhibitor Crude Preparation Purified Preparation
Conc. Act. % I Conc. Act. % I Back.
None 51 0 0.31 0 1.28
PPi 20 43 16 5 0.00 100 0.37
L-Arginine 10 0.15* 61 0.77*
ATP 5 37 ,22 10 0.03* 96 0.87*
AMP 10 73
ADP 10 66
D,L-Valine 20 0.19 39 1.15
L-Valine 10 24 52 .
L-oABA 15 8 84 10 0.21 35 .l.28
Propionate 20 49 4 10 0.21 35 11.28
L—Threonine 20 36 29 10 0.23 28 1.28
KGA 20 47 8 10 0.21 ‘35 1.66
KBA 20 56 -10
L-Canavanine 10 0.25 21 1.28
2-A-3-P 10 0.26 - 19 1.28
MeAsp 10 0.31 0 1.15
L-Lysine 10 0.33 - 5 , 1.03
L-Isoleucine 10 0.34 .- 6 1.25
L-Leucine . 1o 0.3u - 6 ' 1.08

L-Glutamate 10 0.36 -13 1.28

 

* Run with a different enzyme prep. (see Figure 8c). The control
reaction is 98% of uninhibited reaction for arginine and 72%
for ATP.

Abbreviations used in Table VI:
Conc. - Concentration: of the inhibitorM x 103-.

Act. - For the crude preparation - umoles citrulline lost out of
250 umoles (1/6 of assay, see Methods), for purified
preparation - slope of the reaction line (see Figure 7 ).
Concentrations of other reagents as in Methods except
arginine which was 10'2 M.

% I - Percent inhibition.
Back.

Slope of control reaction (see Figure 7 ).

33

Abbreviations used in Table VI (Continued):

PPi - Inorganic pyrophosphate.

AMP - Adenosine 5‘-monophosphate.

ADP - Adenosine 5’-diphosphate.

KGA - o-Ketoglutarate.

KBA - a-Ketobutyric Acid.

2-A-3-P - 2-Amino-3-phosphonopropionic acid.
MeAsp - BAMethylaspartic acid.

by Carbonneau and Berlinguet (32) that L-valine inhibits the con-
version of citrulline to urea in rat liver homogenates.

Because there is considerable argininosuccinase activity in
crude extracts (Table IV) and because argininosuccinate synthetase
is probably reversible (26), when argininosuccinate is added to the
assay, it is probably converted to both arginine and citrulline.
Since arginine interferes with the assay for citrulline, neither
argininosuccinate nor arginine were tested for inhibition in the
crude extract.

Properties of the Purified Preparation. Fractions from the
DEAE cellulose column (Figure 2) exhibited similar argininosuccinate
synthetase activities whether measured by either the colorimetric or
the spectrophotometric assay. Measured spectrophotometrically, this
activity is dependent upon both aspartate and citrulline (Table V).
A magnesium requirement could not be demonstrated using this assay
method since magnesium is required for myokinase and pyruvate

kinase. After a 3 to 5 minute lag, there is some reaction without

34

added ATP. This may be due either to bound ATP in the extract or
the presence of traces of ATP in the reagents.

The addition of inorganic pyrophosphatase and preincubation
with magnesium both cause about a 20 percent increase in activity
(Table V). After a slow start (about 30 sec.), the activity is
linear with respect to time until 2/3 of the NADH is oxidized
(Figure 7). The activity is linear with respect to enzyme
concentration up to at least twice the concentration of enzyme
used in the studies.

The background reaction does not depend upon the addition of
aspartate, citrulline, or ATP (Table V). The rate of this reac-
tion is not linear with respect to either time (Figure 7) or
enzyme concentration (Figure 8a). The activity is, however,
dependent upon the addition of phosphoenol pyruvate (Table V)
so it cannot be explained simply by assuming the presence of an
NADH oxidase. A major part of the activity may be ATPase, although
the omission of ATP causes a alight increase in activity (Table V).
Adenosine triphosphate concentration of 10'2 M inhibits the
reaction about 30 percent (Figure 6). This background activity
is not understood and was not studied further.

The compounds which inhibit argininosuccinate synthetase in
the purified preparation are the same ones which inhibit the
reaction in the crude preparation (Table VI). In addition,
arginine could be tested in the purified preparation and was

found to inhibit strongly (see also Figure 8c). There are some

35

Figure 7. Raw Data from Spectrophotometric Assay

O.D. for Descending Line

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.9- —0.9

1.8 — -0.8

107— -007
o
a
"-1
.4
I:
w-l
'2

1.5 — -O.S 3
2

1.4.. fl —o.u g
1H
d

1.3— ‘0-3 o’

1.2—4 —o.2

1.1 — '-0.1

1.0 \ n

7 6 5 4 3 2 l 0
Minutes

Ascending line was obtained with the reaction cell in the
"Reference" side and the control cell in the "Sample" side
of a Beckman DB Spectrophotometer. The descending line was
obtained by removing the reaction cell and measuring changes
in the control cell only.

36

Figure 8. Factors Affecting Argininosuccinate Synthetase
Measured in the Partially Purified Preparation

8a. Enzyme 8b. Substrates 8c. Inhibitors

   

 

 

 

 

e
44 +4
.,.‘ "-4
> >
'5‘ '5‘
2.0 — o 0
<1 <2
H 'U
m m
U «u
0 'H '94 '
D- 1.) .D .
O a) -.-1
a s a
1.0 -— g 'E'
, 1 D
/ 4H “ 4H
C“ l:
m, m
U U
1' L4 ‘ Ll
/ /' 0 ‘ 0
g: m m
b I 1
0.2 5 10 5 10
m1 Enzyme Substrate M x 103 Inhibitors
M x 103
Filled-in points refer to activity of the control reaction.
8b. Theoretical activity is the V ax obtained from a Lineweaver-
Burk plot of the aspartate ang citrulline data. For ATP,
100 percent activity is the maximum activity when aspartate
and citrulline are 2 x 10‘2 M.
D ATP, + L-citrulline, x L—aspartate.
8c. 0 inorganic pyrophosphate, a L-arginine,

X L-a-aminobutyrate, A 20 mM D,L-valine

37

differences in the degree of inhibition exhibited in the two
assays. Pyrophosphate is a much stronger inhibitor in the puri-
fied preparation and inhibition by propionate is somewhat more
pronounced. It should be remembered that both the specific
activity and the citrulline concentration are quite different

in these two assays.

DISCUSSION

Carbamyl phosphate was proposed as the carbamyl donor in
carbamyl aspartic acid and citrulline synthesis in 1955 (33).
Since then, several different enzymes have been found with the
ability to form active carbamyl groups (34), although there may
be some question of whether or not these enzymes are normally
carbamyl, acetyl, or formyl phosphokinases (35,36). Most organisms
are thought to derive the carbamyl groups used in both arginine and

pyrimidine synthesis from a single source. Escherichia coli is an

 

example of such an organism. Carbamyl phosphate synthesis has been
demonstrated in E. 321; by several workers (37,38,39). A single
gene mutant, P678B1, which lacks this activity requires both
arginine and pyrimidine for growth. Reversion of P678Bl to
prototrophy is a single genetic event (39).

Neurospora crassa, on the other hand, appears to have two
sources of carbamyl groups -- one specific for pyrimidine synthesis
and the other specific for the synthesis of arginine (7,12). This
conclusion is drawn from attempts to explain the pyr-3a mutation.

Pyr-3a mutants will, of course, grow when pyrimidines are added
to the culture medium. But they vdll also grow under a variety of
other conditions which.fall into two general groups: (1) condi-
tions which reduce the intracellular concentration of arginine,
and (2) conditions which increase the availability of carbon
dioxide. In the first case, lowered arginine concentrations

presUmably derepress the arginine-specific carbamyl source so that

38

39

the extra carbamyl groups formed can be used in the pyrimidine
pathway. In the second case, an increase in carbon dioxide con-
centration apparently stimulates the production of carbamyl groups
by a mass action effect. In both cases, however, the carbamyl
groups which go into pyrimidines are thought to come from the
arginine-specific enzyme since the addition of very small amounts
of arginine restores the pyrimidine requirement (2,7,10).

The theory provides a plausible explanation for experiments
with pyr-3 mutants and is further supported by experiments with
other mutants. A situation analogous to pyr-3a suppression has
been reported for two unlinked arginine mutants -- arg-Z and
arg-3 (40,41). In this case, the arginine requirement is removed
by a mutation in the structural gene for aspartic transcarbamylase.
The erg-2 and erg-3 loci are thought to be concerned with the
formation of arginine-specific carbamyl groups. The reduced
pyrimidine production apparently derepresses the pyrimidine—specific
carbamyl source.

But the most direct evidence for the existence of two carbamyl
sources comes from studies with the carbamyl-forming system it-
self (21). Extracts from wild type strains incorporate radioactive
bicarbonate into the ureido carbon of citrulline when incubated
with ammonia, ornithine, ATP, and magnesium. This "carbamyl phos-
phokinase" activity is not present in the erg-3 mutants. If there
are, indeed, two carbamyl sources in N. crassa, one might expect

the arg-3 mutants to exhibit activity from the pyrimidine-specific

source. Although this pyrimidine-specific enzyme was not detected
by this method, its existence can be inferred from the observation
that the double mutant, pyr-l arg-3, still accumulates carbamyl
aspartate. The fact that arg-2 exhibits carbamyl phosphokinase
activity may mean that there are two steps in the formation of
active carbamyl groups in the arginine pathway.

Even though only one of the two proposed carbamyl sources has
been clearly demonstrated, for the rest of the discussion I will
assume the theory is correct. It is not even certain that carbamyl
phosphate is the carbamyl donor, even though it is an effective
substrate in yitgg, Nevertheless, I will call the carbamyl donor
"carbamyl phosphate" and the enzyme or enzymes involved "carbamyl
,phosphokinase."

My first contribution toward explaining the action.of pro-
pionate and o-aminobutyrate was the demonstration that these
compounds reduce the intracellular arginine concentration. To
this extent, they imitated the suppressor and provided a separate
body of evidence for the theory just presented. This reduction
in arginine gave a partial answer to the original problem and
immediately raised another question -- how'do propionate and
o-aminobutyrate lower the concentration-of arginine?

The arginine concentration might be lowered either by inhibit—
ing arginine synthesis or by increasing its removal. I chose to
concentrate on conditions which might inhibit synthesis. Since the

suppressor reduces ornithine transcarbamylase activity, a reasonable

41

starting point was to determine whether or not propionate and o—
aminobutyrate inhibit this enzyme.

Although high concentrations of o-aminobutyrate did inhibit
ornithine transcarbamylase, this inhibition was not considered a
sufficient explanation for the drastic reduction in arginine
concentration (42). Not only were propionate and o-aminobutyrate
poor inhibitors, but their presence in the growth medium caused
derepression of ornithine transcarbamylase to more than three
times the normal activity.

It is not likely that the arginine specific carbamyl phospho-
kinase is inhibited or repressed by the growth-promoting compounds
since this enzyme is necessary to supply carbamyl phosphate for
both pyrimidine and arginine synthesis.

If we disregard for a moment the possibility that propionate
and o-aminobutyrate inhibit the synthesis of aspartate or orni-
thine, there are only two enzymes which remain to be considered --
argininosuccinate synthetase and argininosuccinase. The finding
of Fairley and Adams (10) that citrulline is a poor inhibitor of
growth on o-aminobutyrate, while arginine is an extremely potent
inhibitor, supports the possibility that the conversion of
citrulline to arginine does not readily occur in the presence of
a-aminobutyrate.

Fincham and Boylen (23) have shown that the arg-lO mutant,
which has lost argininosuccinase activity, accumulates arginino-

succinate when grown on arginine. Similarly, the arg-l arg-lO

42

double mutant, which is also deficient in argininosuccinate synthetase,
accumulates citrulline (25). If growth of 1298 on a—aminobutyrate
involves the inhibition of one of these enzymes, similar increases

in either argininosuccinate or citrulline might be expected.

In the experiments described here, no accumulation of arginino-
succinate could be detected. Since there was a considerable con-
centration of citrulline under normal growth conditions, the results
were not sufficiently precise to determine whether or not the
citrulline concentration did increase. But the ornithine concen-
tration did go down when the mold was grown on a-aminobutyrate.

This relative increase in citrulline concentration with respect to
arginine and ornithine may be all that can be expected. It should
be remembered that the reduction in arginine synthesis caused by
o-aminobutyric acid is considerably different from the complete
blocks described by Fincham and Boylen and Newmeyer.

In the first place, growth on o-aminobutyrate does.not cause
a complete block in arginine synthesis. There is some flux
through the pathway from citrulline to arginine. In the second
place, and perhaps more important, organisms grown on o—amino-
butyrate were not fed arginine. A large part of the citrulline
which accumulated in the work.described.by Newmeyer may have come
by way of ornithine from the arginine which was included in the
growth medium.

The possible role of ornithine in carbamyl phosphate avail-

ability has never been discussed in print. The strong growth

43

inhibition caused by adding small quantities of arginine may, in
part, be due to formation of ornithine which can compete with
aspartic acid for the available carbamyl phosphate as well as to
repression of carbamyl phosphokinase. This possibility is
consistent with the observation of Fairley and Adams that growth
on o-aminobutyrate is much more strongly inhibited by ornithine
than by citrulline.

A study of argininosuccinate synthetase finally gave clear
evidence that at least part of the reduction in arginine concen-
tration is due to inhibition of this reaction. Both o-amino-
butyrate and propionate inhibit partially purified arginino-
succinate synthetase in 33359. The fact that propionate was a
much less effective inhibitor in the crude system was probably
due to its rapid removal by other enzymes in the crude extract.
Not only do propionate and o-aminobutyrate inhibit but so does
L-valine, an amino acid which appears to accumulate in cells
grown on either of the other two inhibitors.

So far, evidence has been presented indicating that propionate
and abaminobutyrate inhibit argininosuccinate synthetase, both
directly and indirectly. Other unsuspected effects of these
compounds may well exist. For example, the possibility that they
inhibit the formation of ornithine from glutamic acid, either
directly or indirectly, has not been excluded.

The metabolic changes which have been discussed in this

thesis emphasize the fact that an organism is a unit and not

44

just a collection of independent metabolic pathways. The addi-
tion of o-aminobutyrate to the growth medium of N. crassa affects
the synthesis of valine, isoleucine, arginine, and pyrimidine. I
have only considered the most obvious changes in pyrimidine and
amino acid metabolism. It is not unlikely that many other changes
occur as the cell adjusts to the introduction of a gratuitous
metabolite. A thorough study of these interactions is beyond the
scope of this study.

In summary, the primary metabolic defect in the pyr-3a mutants
of N. crassa is apparently the inability to provide a pyrimidine-
linked source of carbamyl phosphate. Derepression and reduced
consumption of the arginine-linked carbamyl source allows growth
by providing excess carbamyl phosphate, some of which can be used
for pyrimidine synthesis. Although some details remain to be
examined more fully, the experiments described in this thesis pro-
vide a coherent explanation for the growth—promoting properties of
propionate and o-aminobutyrate. ‘Gnowth of N. crassa on.either of
these compounds is accompanied by a low concentration of intra-
cellular arginine, presumably-leading to derepression of carbamyl
phosphate synthesis. The low arginine level is due, at least in
part, to the direct inhibition of argininosuccinate synthetase by
propionate and o-aminobutyrate. These compounds may also reduce
argininosuccinate synthetase activity indirectly by stimulating
the production of valine which also inhibits the reaction. The
discovery that argininosuccinate synthetase is inhibited by arginine
may have an important bearing on the control of arginine synthesis

in this organism.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

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