(CANCER RESEARCH 31, 985—991, July 1971]
Rate-limiting Steps in the Interconversion of Purine
Ribonucleotides in Ehrlich Ascites Tumor Cells in Vitro'
G. W. Crabtree2 and J. Frank Henderson3
University ofAlberta
Cancer Research Unit (McEachern Laboratory) and Department of Biochemistry, Edmonton, Alberta, Canada
SUMMARY
The conversion of inosinate to guanylate in Ehrlich ascites
tumor cells incubated in vitro in Krebs-Ringer phosphate
medium is limited first by the concentration of glutamine and
then by the concentration of inosinate. The conversion of
inosinate to adenylate is limited by the concentration of
aspartate. Adenylate conversion to guanylate is limited first by
the concentration
of glutamine and then probably by
adenylate
deaminase activity. Guanylate
conversion to
adenylate is limited by guanylate reductase activity.
INTRODUCTION
Purine ribonucleotides are interconverted by a system of 6
enzymes arranged in 2 cycles which have a common
intermediate in inosinate:4
adenylate
guanylate
\
/\N7h
@
Adenylosuccinate
inosinate
aspartate
+
\+
xanthylate
NAD
These reactions may at least potentially be regulated by the
amounts of the enzymes involved, by the concentrations of
the nucleotide intermediates and of coenzyme and amino acid
substrates, and by allosteric activation and inhibition.
The relative activities of several enzymes of purine
ribonucleotide interconversion have been measured by McFall
and Magasanik (7) in extracts of L-cells and of Ehrlich ascites
tumor cells. If total enzyme activities were rate limiting, one
I This
work
was
supported
by
the
National
Cancer
Cancer
Institute
Institute
of
Canada.
2 Research
Fellow
of
the
National
of
4The enzymes of purine ribonucleotide interconversion are:
@
dehydrogenase
(IMP:NAD oxidoreductase,
for example,
has already been shown to be limiting for protein
synthesis (8) and for purine biosynthesis de novo (4), and
Hershko et a!. (6) have proposed that the availability of this
amino acid may also limit guanylate synthesis in rabbit
erythrocytes in vitro. Finally, Fontenelle and Henderson (3)
have suggested that intracellular concentrations of aspartate
may be limiting for adenylate synthesis from inosinate.
Numerous studies (reviews in Refs. 1 and 12) have also
shown that most of the enzymes of purine ribonucleotide
interconversion are activated or inhibited by one or another
purine nucleotide.
Although these reactions and their
regulations have been studied individually in some detail in cell
extracts and with partially purified enzymes, relatively little
work has been done to elucidate the controls of these
reactions as they operate as an integrated system in intact
cells. In this study, the rate-limiting steps in the pathways of
purine ribonucleotide interconversion in Ehrlich ascites tumor
cells have been identified under several conditions of
incubation in vitro.
This study has been greatly facilitated by the development
of procedures for the rapid analysis of radioactivity in purine
ribonucleotides, ribonucleosides, and bases in large numbers of
small samples; these methods are given in detail.
Canada.
Present address: Division of Biological and Medical Sciences, Brown
University, Providence, Ri.
3To whom inquiries should be addressed.
inosinate
would conclude from their results that the synthesis of
adenine nucleotides from inosinate would take place much
more readily than the synthesis of guanine ribonucleotides.
Furthermore,
the rate-limiting reactions for these two
processes would be adenylosuccinate
lyase and inosinate
dehydrogenase, respectively. Santos et a!. (10) also suggested
that inosinate dehydrogenase
activity might limit the
conversion of adenylate to guanylate in rat brain extracts.
Rates
of
interconversion
of adenine
and guanine
ribonucleotides are slow in rabbit erythrocytes in vitro (6),
and activities of adenylate deaminase and guanylate reductase
may be limiting.
However, it is uncertain whether substrate and cofactor
concentrations for these enzymes are saturating in Ehrlich
ascites tumor cells in vitro. The concentration of glutamine,
MATERIALS AND METHODS
EC 1.2.1.14];
guanylate synthetase (xanthosine-5'-phosphate ligase (AMP), EC
6.3.4.1] ; guanylate reductase (reduced NADP:GMP oxidoreductase
(deaminating),
EC 1.6.6.8] ; adenylosuccinate synthetase
(IMP:L-aspartate ligase (GDP), EC 6.3.4.4] ; adenylosuccinate lyase
(adenylosuccinate AMP lyase, EC 4.3.2.2] ; adenylate deaminase (AMP
aminohydrolase, EC 3.5.4.6].
Received December 11, 1970; accepted March 5, 1971.
4 C
(49.5
mCi/mmole),
(52.6
and
mCi/mmole),
4 C
C (31 .7 mCi/mmole)
were obtained from Schwarz BioResearch, Inc., Orangeburg,
N. Y.; purine bases and ribonucleosides were from Sigma
Chemical Company, St. Louis, Mo.; purine ribonucleotides
were from P-L Biochemicals, Milwaukee, Wis.; L-glutamine
JULY 1971
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985
G. W. Crabtree and J. Frank Henderson
@
@
was from Calbiochem, Los Angeles, Calif. ; and L-aspartic acid
was from Mann Research Laboratories, New York, N. Y.
Hadaci di n
(N-formylhydroxyaminoacetic
acid)
and
6-diazo-5-oxo-L-norleucine
were gifts of the Cancer
Chemotherapy
National Service Center, National Cancer
Institute, Bethesda, Md.
Six days after i.p implantation in ICR Swiss mice of
approximately
106 Ehrlich ascites tumor cells, cells were
removed and washed 3 times with buffered saline (140 mM
NaCl:lO mM Tris buffer, pH 7.4:4 mM sodium phosphate
buffer, pH 7.4) containing 5.5 mM glucose. A 2% cell
suspension was incubated in modified Krebs-Ringer phosphate
medium ( I 10 mM NaCl:4.9 mM KC1:1.2 mM MgSO4 :25 mM
sodium phosphate buffer, pH 7.4) containing 5.5 mM glucose
and other additions as required. All incubations were carried
out in a water bath at 37°with shaking at 80 oscillations/min
and air as the gas phase. In all experiments, cells were
incubated with glucose alone for 20 mm before radioactive
precursors were added. Preliminary experiments had shown,
that prior incubation with glucose in a high-phosphate medium
gave maximal rates of purine ribonucleotide synthesis from
purine bases.
After various periods of incubation, 0.5-ml samples of
incubation media containing cells were transferred to tubes
containing 25 p1 of cold 4.2 M perchioric acid; acid extracts
were subsequently neutralized with 25 @.zl
of 4.42 N KOH.
After centrifugation , samples were chromatographed.
Baker-Flex polyethyleneimine cellulose thin layers on Mylar
sheets (Fisher Scientific, Edmonton, Alta., Canada) were
used to separate purine ribonucleotides by I -dimensional
chromatography
in a modification
of the method of
Randerath and Randerath (9). Sheets were first developed for
5 hr with 4 M sodium formate buffer, pH 3.4, dried, and then
developed overnight with methanol:water (1 : 1). After drying,
10 or 20 @zl
of cell extract plus about 30 nmole of each purine
ribonucleotide carrier were applied as a I -cm streak 2 cm from
the bottom of the sheet. A wick of Whatman No. 3MM paper
was stapled to the top of the sheet, and it was developed
overnight with methanol:water
(1 : 1) to wash salts, purine
bases, and ribonucleosides onto the paper wick; the wick was
then discarded. For separation of the ribonucleotides, the
sheets were developed with increasing concentrations
of
sodium formate buffers, pH 3.4, as follows: 0.5 M formate
buffer to a line 2.5 cm above the origin, then 2.0 M formate
buffer to a line 7.0 cm above the origin, and finally 4.0 M
formate buffer to the top of the plate. The sheets were dried,
and nucleotide-containing
areas were visualized under UV
light. With this method, the following nucleotides were well
separated (the distance of each from the origin is given in cm):
GTP (1 .3), ATP (3.5), GDP (4.5), ADP (9.0), GMP (10.0),
XMP (1 1.0), IMP (12.3), AMP (14.0), and NAD (15.5). Eight
samples were usually analyzed per sheet.
Eastman Kodak unsubstituted cellulose thin layers on Mylar
sheets (Fisher Scientific) were used to separate purine bases
and ribonucleosides by 2-dimensional chromatography. Each
sheet was developed in the first direction for about 50 mm
with acetonitrite:0.l
M ammonium acetate, pH 7.0:ammonia
(60:30: 10). After the plates were dried, areas below the origin
(which
986
was
2.5 cm
in each dimension
from
1 corner) and 3 cm
from the top were scraped off and discarded. The plates were
rotated 90° and developed in the 2nd direction with
1-butanol: methanol:water:ammonia
(60:20:20: 1);
after
drying, the 2nd dimension was redeveloped with the same
solvent. Purine-containing areas were visualized with UV light.
With this method, the following compounds were well
separated (the distance of each from the origin is given in cm
with the 1st dimension followed by the 2nd dimension):
adenine (10.8, 11.7), adenosine (13.2, 10.0), hypoxanthine
(10.6, 7.8), guanine (6.9, 5.8), inosine (12.3, 6.7), guanosine
(10.9, 5.7), xanthine (6.9, 4.7), xanthosine (1 1.3, 4.5), and
uric acid (5.6, 3.2). Nucleotides remained as a streak along the
line of 1st development.
UV-absorbing areas of the chromatography sheets were cut
out and placed in counting vials, phosphor solution was added
(4 g PPO and 0.1 g POPOP per liter of toluene), and
radioactivity measurements were made at 72% counting
efficiency.
Results presented below are measurements of the amounts
of radioactivity in each metabolite, expressed as nmoles/g of
cells, rather than the total amount of each metabolite. Average
values
from
duplicate
samples
are reported.
The results
are
representative of those obtained in at least 2 experiments.
RESULTS
Factors that are rate limiting for the conversion of inosinate
to adenylate and guanylate were studied first. The data
presented in Chart 1 show that Ehrlich ascites tumor cells in
vitro converted
C more extensively to
adenine nucleotides
(including NAD) than to guanine
LI
w
90
I-.
0
LU
-j
L)
:@ 60
z
0
U.
0
30
z
J.
LU
L)
I
LU
a-
_0
30
60
90
MINUTES
Chart 1. Relative incorporation of
anin4
C into adenine
and guanine nucleotides. Ehrlich ascites tumor cells, 2% by volume,
were incubated in 25-mi Erlenmeyer flasks at 37°with shaking with an
atmosphere
of air in 5.0 ml of Krebs-Ringer
medium
containing
25 mM
sodium phosphate buffer, pH 7.4, and 5.5 mM glucose. After 20 mm,
hypoxanthine-' 4C was added to final concentrations of 5 MM(o, i@)or
100 jsM (., £).At various times, portions were removed for analysis of
radioactivity in adenine nucleotides (., o) and guanine nucleotides (a,
a').Each point representsthe mean of separateanalyse@of duplicate
flasks in I experiment; the results are representative of those obtained
in 4 experiments.
CANCER RESEARCH VOL. 31
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1971 American Association for Cancer Research.
Purine Ribonucleotide
nucleotides
precursor
(plus xanthylate).
(5 pM),
the
ratio
With a low concentration
of incorporation
into
of
a-
adenine
LI
nucleotides relative to that into guanmne nucleotides was
approximately 3 after 30 mm incubation, whereas this ratio
was approximately
6 at the same time when the initial
extracellular concentration of hypoxanthine was 100 jiM.
Further studies were conducted to identify the rate-limiting
steps and factors on the pathway of guanylate synthesis from
inosinate. Inosinate or xanthylate might be expected to
accumulate
if inosinate
dehydrogenase
or xanthylate
LU
I-
nucleotides
might
be dephosphorylated
LU
LI
z
0
precursor
@
@
@
concentration
S jiM
the
incorporation
of
radioactive
inosine
formation
in
cells
10
U.
0
I—
5
z
LU
LI
if they
LU
a-
0-
0
30
60
incubated
with hypoxanthine-' C or
C do not necessarily measure the
possible rate-limiting character of IMP dehydrogenase because inosine
may be synthesized by pathways alternative to dephosphorylation of
inosinate. Thus, inosine may be made directly from hypoxanthine by
purine nucleoside phosphorylase and from adenine via adenylate and
adenosine. At the present time, the relative rates of the alternative
pathways involved have not been evaluated.
90
MINUTES
Chart 2. Formation of radioactive inosinate and xanthylate from
hypoxanthine-' 4C. Tumor cells were incubated as described in Chart 1
with 100 @LM
hypoxanthine-' 4C. Portions were removed at various
times for analysis of inosinate (.) and xanthylate (0).
aLI
0
I—
U.
0
I-
z
LU
LI
LU
a-
of
radioactivity into guanmne compounds was greater than that
into adenine compounds, although the rate of incorporation
into guanine compounds decreased more rapidly than did the
other process. After 90 mm of incubation, the ratio of
radioactivity in adenine compounds relative to that in guanine
compounds was about 1:3. When the initial extracellular
concentration of
C was 100 jiM , the rates of
its incorporation
into adenine compounds and guanine
compounds were similar for the first 30 mm of incubation. At
later times, the rate of incorporation into adenine compounds
decreased relative to that into guanine compounds.
The rate-limiting
character
of xanthylate
aminase
demonstrated above might have been due to the total activity
of this enzyme or to the concentration of another substrate of
this reaction, glutamine. The effects of addition of this amino
S Measurements
@
of
15
I—
aminase,
began to accumulate, the formation of nucleosides and bases
by cells incubated with hypoxanthine-'4C
was measured.
After 90 mm of incubation, 1190 nmoles/g cells of radioactive
xanthosine plus xanthine accumulated under these conditions;
this amounted to almost 30% of the total acid-soluble
radioactivity present in the sample. It will be shown below
that the main pathway of xanthosine and xanthine synthesis
from hypoxathine in these cells is via xanthylate rather than
by xanthine
oxidase action on hypoxanthmne. These
observations
both imply that significant amounts of
xanthylate were very readily dephosphorylated.
Xanthylate
aminase, therefore, appears to limit the conversion of inosinate
to guanylate under these conditions.5
It thus became apparent that the flow of radioactive
compounds along the mnosinate-guanylate pathway could not
be accurately estimated by measurements of radioactive
nucleotides only. Chart 3 shows the relative distribution of
radioactivity in “adeninecompounds― (adenine nucleotides,
NAD, and adenosine) and in “guaninecompounds― (guanmne
nucleotides, xanthylate, xanthosine , guanosine, xanthine , and
guanine) after incubation of cells with
pothi4
C. At a
20
0
respectively, were rate limiting. However, Chart 2 shows that
the concentrations of radioactive inosinate and xanthylate
remained very low throughout the incubation period.
Because
Interconversions
MINUTES
Chart 3. Relative conversion of hypoxanthine-' C to “adënine
compounds― and “guaninecompounds― (see text). Tumor cells were
incubated as described in Chart 1 with 5 MM(o, @)or 100 @iM
(., a)
hypoxanthine-' 4C. Portions
@reremoved at various times for analysis
of adenine compounds (., o) and guanine compounds (a, @).
acid to incubation media on the synthesis of guanine
nucleotides and of xanthosine plus xanthine were therefore
studied. The accumulation of radioactive xanthosine plus
xanthine after 90 mm of incubation decreased from 1210
nmoles/g in the absence of glutammne to 74 nmoles/g in the
presence of 2 mM glutamine. (These data also show that
almost
no xanthine
is being
formed
via xanthine
oxidase
activity on hypoxanthine under these conditions.) As would
be expected, the incorporation of
C into
guanine nucleotides was markedly increased in the presence of
glutamine (Chart 4).
Further information regarding the role of glutamine in
regulating xanthylate aminase activity came from studies with
diazooxonorleucine, an antimetabolite of glutamine, and with
methionine sulfoximine, an inhibitor of glutamine synthetase.
Chart S shows that the concentration of diazooxonorleucine
used almost completely inhibited the synthesis of guanine
JULY 1971
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987
G. W. Crabtree
and J. Frank Henderson
The decrease in the amount of radioactivity found in
xanthosine plus xanthine when glutamine was added was not
equaled by the increase in the radioactivity found in guanine
nucleotides under the same conditions. This discrepancy was
due in part to a doubling in the amount of radioactive
225
0
150
guanosine
‘I)
LU
-I
plus guanine
0
C
4C
75
This
converted
apparent
phosphoribosyltransferase
0
)
30
60
90
MINUTES
@
formed.
However,
this amounted
to
only I to 2% of the total radioactivity involved. Another cause
of the discrepancy was a marked decrease in the amount of
Chart 4. Effect of glutamine on the synthesis of radioactive guanine
nucleotides from hypoxanthine-' C. Tumor cells were incubated as
described in Chart 1 with 100 pM hypoxanthine-' 4C with (0) and
without (.) 2 mM glutamine.
to
ribonucleotides
decrease
activity
in
(Chart
6).
hypoxanthine
may be caused by diversion
of phosphoribosylpyrophosphate
to the pathway of purine
biosynthesis de novo, which is still operating to some extent
even at 100 jiM hypoxanthine (4).
Chart 7 shows another alteration in purine metabolism upon
the addition of glutamine. A considerable amount of
radioactive inosinate, which in the absence of glutamine would
have been converted to xanthosine and xanthine, was in the
presence of this amino acid converted to adenine nucleotides
rather than to guanine nucleotides. Although the mechanism
1@
,
I
100
@
4200
@
0
!@
2800
75
‘I,
LU
@1
0
50
C
00oo@
25
@
1400
0
30
60
90
0
MINUTES
@
0
Chart 5. Effect of diazooxonorleucine on the synthesis of radioactive
guanine nucleotides from hypoxanthine-' 4C. Tumor cells were
incubated as described in Chart 1 with 100 @M
C with
(0)
and
without
(.)
35
@M diazooxonorleucine.
Table 1
Effect ofmethionine sulfoximine on the synthesis of
radioactive xanthine plus xant ho sine
from hypoxanthine-' 4C
Tumor cells were incubated as described in Chart 1 with 100
hypoxanthine with and without 5 mM methionine sulfoximine.
AdditionsIncubation
(nmoles/g)None
time
(mm)Xanthine
I
I
60
90
MINUTES
Chart 6. Effect of glutamine on the utilization of
nm4
Tumor cells were incubated as described in Chart 1 with 100
hypoxanthine-' 4C with (o) and without (.) 2 mM glutamine.
I
I
I
I
30
60
90
C.
@M
I
1500
@M
plus
xanthosine
I
30
0
‘I,
LU
-I
1000
0
C
Methioninesulfoximine30
60
30
60750
978
960
1242
@
500
0
C
@
nucleotides from
C. The data in Table 1 show
that the formation of radioactive xanthine plus xanthosine was
also increased when glutammne synthesis was inhibited by
methionine sulfoximine. (Glutamine completely overcame the
effect of this amino acid analog.)
988
MINUTES
Chart 7. Effect of glutamine on the synthesis of radioactive adenine
nucleotides from
hi4
C. Tumor cells were incubated as
described in Chart 1 with 100 @zM
hypoxanthine-' 4C with (o) and
without (.) 2 mM glutamine.
CANCER RESEARCH VOL.31
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1971 American Association for Cancer Research.
Purine Ribonucleotide
@
@
Interconversions
of this stimulation of adenine nucleotide synthesis will be
discussed below, this observation implies that in the presence
of glutamine total inosinate dehydrogenase activity was still
not rate limiting for the conversion of inosinate to guanylate.
Instead, the amount of inosinate available to this enzyme in
the face of increased adenylosuccinate synthetase activity
appeared to be the more important regulating factor.
If adenylosuccinate
synthetase did have a competitive
advantage over inosinate dehydrogenase with respect to
utilization of inosinate, then inhibition of the former enzyme
with hadacidin ( 11) might alter this situation. Chart 8 shows
that, although this analog of aspartate did inhibit the synthesis
of radioactive adenine nucleotides by more than 80%, there
high initial extracellular concentrations of aspartate were used,
but the intracellular concentration attained has not been
determined. Chart 9 shows that the addition of aspartate
increased the conversion of
an4
C to adenine
nucleotides
almost 2-fold, whereas guanine nucleotide
synthesis was scarcely affected. The synthesis of xanthine plus
xanthosine decreased from 1190 to 876 nmoles/g after 90 mm
of incubation, suggesting again that inosinate was diverted
away from the pathway of guanylate synthesis in the presence
of aspartate.
Although
radioactivity
in adenylosuccinate
was not
routinely
measured
in these experiments,
preliminary
experiments have shown that it does not appear to accumulate
was little
in the
or no stimulation
of the synthesis
of radioactive
guanine nucleotides. It would be expected, however, that most
of the product of the inosinate dehydrogenase reaction would
accumulate
as xanthosine
plus xanthine
under these
conditions; after 90 mm of incubation, accumulation of these
products increased from 1254 to 1490 nmoles/g in the
presence of hadacidin. An increase in inosine formation, as
well as an apparent decreased utilization of
C,
were also observed in the presence of hadacidin. These changes
might be due, at least in part, to increased dephosphorylation
of inosinate ; whether this increased dephosphorylation is due
to accumulation of inosinate consequent upon saturation of
inosinate dehydrogenase with this substrate, or simply to
dephosphorylation of increased amounts of inosinate without
saturation of inosinate dehydrogenase, is not clear.
The stimulation of radioactive adenine nucleotide synthesis
from hypoxanthine-' 4C upon addition of glutammne to
incubation media (Chart 6) was probably due to the rapid
conversion of this amino acid to aspartate, a substrate of
adenylosuccinate synthetase; this process has previously been
shown to occur in these cells (5). Aspartate itself was therefore
added
to
incubation
media
and
its effects
on
presence
or absence
for adenylate
Adenylosuccmnate
synthesis
from inosinate.
When
C was used as substrate of nucleotide
synthesis in Ehrlich ascites tumor cells, almost 95% of the
total nucleotide fraction was composed of adenine nucleotides
themselves. Even when its conversion into bases and
nucleosides not containing adenine was measured (Table 2), 89
I
I
2100
0
..%.
1400
LU
0
C
700
U
mn4 C metabolism were measured. Becausethe
0
30
cells are not very permeable to dicarboxylic amino acids (2),
1200
of aspartate.
synthetase would appear therefore to be the rate-limiting step
60
90
MINUTES
Chart 9. Effect of aspartate on the synthesis of radioactive adenine
and guanine nucleotides from hypoxanthine-' 4C. Tumor cells were
incubated as described in Chart 1 with 100 @M
hypoxanthine-' 4C with
-
(0,
i@@)and
without
(.,
a)
20
mM
aspartate.
Portions
were
removed
at
various times for analysis of adenine nucleotides (., o) and guanine
nucleotides (a, is).
0800
-
LU
Table2
Conversion
‘C into metabolites
not containing adenine°
Tumor cells were incubated as described in Chart 1.
-I
0
@
@
C 400
-
Adenine-'4C
concentration
radioactivity)2010
(NM)Incubation
0@-@
MINUTES
14.25010
Chart 8. Effect of hadacidin on the synthesis of radioactive adenine
and guanine nucleotides from hypoxanthine-' 4C. Tumor cells were
incubated as described in Chart 1 with 100 @M
hypoxanthine-1 4C with
(0,
t@) and
without
(5,
a)
100
@g/ml of
hadacidin.
Portions
(mm)Metabolites
(% total
307.2
303.4
302.5
5.5
were
removed at various times for analysis of adenine nucleotides (., o) and
guanine nucleotides (a, ‘s).
6.710010
not
containing adeninea
time
a Inosinate,
xanthylate,
guanine nucleotides,
hypoxanthine,
inosine,
xanthine, xanthosine, guanine, and guanosine.
JULY 1971
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989
G. W.CrabtreeandJ. Frank Henderson
@ to 97% of the total radioactivity was in adenine and related
nucleosides and nucleotides.
Table 3 shows that neither radioactive inosinate nor
radioactive xanthylate accumulate in cells incubated with
adenine-'4C, but 221 nmoles/g of radioactive xanthosine plus
xanthine accumulated after 90 mm of incubation with 100
mM adenine-'4C. Other experiments have shown that this
accumulation
did not occur in the presence of added
glutamine and that the amount of radioactive guanine
nucleotides was concomitantly increased. Some inosine and
hypoxanthine were formed both in the presence and absence
of glutamine, but, because these could be formed via the
dephosphorylation of adenylate as well as by that of inosinate,
rate-limiting steps could not be evaluated.
An experiment was done with adenine-'4C to determine
whether any radioactive inosinate formed from adenylate was
converted back to adenylate via adenylosuccinate synthet.ase.
The conversion of radioactivity from adenine into inosine was
increased from 74 to 102 nmoles/g after a 90-mm incubation
of hadacidin. If we assume that hadacidin has no effect on the
dephosphorylation
of adenylate and deamination of adeno
sine, it may tentatively be concluded that the increased
amount of radioactive inosine was derived from inosinate
which was not utilized by adenylosuccinate synthetase in the
presence of this inhibitor.
When Ehrlich cells were incubated
in vitro with
guanine-' 4C, less than 10% of the precursor was converted to
compounds that did not contain the guanine moiety per se
(Table 4). (Xanthine, a possible catabolite of xanthylate, was
not included by these figures because it may also be formed by
Table3
Concentrations of radioactive inosinate and xanthylate
synthesized from
@4
C
Tumor cells were incubated as described in Chart 1.
the action of guamine deaminase on the precursor guanine-' C
as well as by the catabolism of guanine nucleotides.)
It is apparent that the conversion of guanylate to adenylate
took place very slowly in these cells. Because neither imosinate
nor hypoxanthine
plus inosine accumulated under these
conditions, the rate-limiting step in this process appears to be
guanylate reductase.
DISCUSSION
It is apparent from these studies that the flow of material
along the various pathways of purine ribonucleotide
interconversion cannot be accurately gauged by measurement
of radioactivity in the ribonucleotide
intermediates and
products only. The nucleosides and bases derived from these
compounds may contain significant amounts of radioactivity,
which may not only influence conclusions
regarding
identification of rate-limiting steps but may also change
markedly depending on experimental conditions.
The conversion of inosinate both to guanylate and to
adenylate in cells incubated in this salts:glucose medium was
limited primarily by the intracellular concentrations of the
amino acid substrates of these reactions, glutamine and
aspartate, respectively. No firm evidence was obtained to
indicate that inosinate concentrations ever rose to the point
where total inosinate dehydrogenase activity became rate
limiting.
Instead,
inosinate
appeared
to be either
dephosphorylated
adenylate.
time
cells
(nmoles/g)
2.030
10
3.050
2.530
10
4.0100
1030
10
15Table
4.0
5.0
7.5
12.5
20
25
which
showed
that
previous
suggestions
found
to
distinguish
guaninea(@zM)
radioactivity)20
Incubation
containing
2.630
10
2.7100
2.230
10
2.4
adenine
nucleotides,
990
(3) that aspartate
concentrations
the
dephosphorylation
of
study of regulatory
factors in this
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6.850
a Inosinate,
were
(% of total
5.630
10
adenine, adenosine, and xanthosine.
concentrations
intact cell system.
Metabolites
time
(mm)
glutamine
between
are being begun for further
C
and
adenylate and that of inosinate.
Studies to evaluate the possible regulation of the enzymes
of purine ribomucleotide interconversion by variation in the
concentrations of purine ribonucleoside di- and triphosphates
4Conversion
metabolitesnot
ofguanine-'4C into
a14
containing guanine
notconcentration
adenylosuccinate
might limit adenylosuccinate synthetase activity.
Although no evidence was obtained to indicate that total
activities of inosinate dehydrogenase or of adenylosuccinate
synthetase were rate limiting, some evidence does support the
idea that total activities of adenylate deaminase and guanylate
reductase
may be of greater regulatory
significance.
Unfortunately, this point may remain unclear until means are
Inosinate
(mm)
to
The results of supplementation
with glutammne and the
effects of diazooxonorleucine and methionine sulfoximime are
in agreement with previous studies with Ehrlich ascites tumor
support
(nmoles/g)20
converted
limiting for other processes as well (2—5,8). These results also
Incubationconcentration
14 c
Xanthylate(NM)
or
xanthylate,
hypoxanthine,
inosine,
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991
Rate-limiting Steps in the Interconversion of Purine
Ribonucleotides in Ehrlich Ascites Tumor Cells in Vitro
G. W. Crabtree and J. Frank Henderson
Cancer Res 1971;31:985-991.
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