The Incorporation of Glycine—2-C14into Acid-soluble
Nucleotide Purines*t
MARY P. EDMONDS AND G. A. LEPAGE
(McArd1e MemorieALaboratory, University of Wisconsin, Madison 6, Wi..)
Acid-soluble nucleotides
have recently been
strongly implicated as precursors of polynucleo
tides. Several workers using glycine-2-C'4 (7, 16)
and adenine-C'4 (2, 10, 19, 21) have reported the
rapid turnover of acid-soluble adenine with respect
to nucleic acid adenine in a variety of biological
carcinoma
systems.
(17). The powdered tissues were then extracted
with cold 0.4 M perchloric acid (PCA).3 The ex
tracts were homogenized to facilitate extraction,
and the cold homogenates were centrifuged. The
acid-insoluble
residues were washed twice with
cold 0.2 M PCA, and the washings were added to
the original extract. The clarified extracts were
then neutralized
carefully
with concentrated
KOH. The precipitated potassium perchlorate was
removed, and the extracts were stored at —10°C.
until they were chromatographed.
In experiments
carried
be formed quite directly from this acid-soluble
pool (14). LePage (16) has shown that the in vifro
incorporation
of glycine into the nucleic acids of
mammalian liver and tumor cells could be marked
ly reduced by the addition of unlabeled exogenous
purmnes and their nucleoside and nucleotide de
rivatives.
Our aim in these experiments was to establish
certain time relationships between the incorpora
tion of glycine into the purmnes of the acid-soluble
nucleotides and into the nucleic acids, and to use
this information to select time intervals for further
study which would be most likely to reveal the
existence of nucleic acid precursors in the acid
soluble extracts after the injection of glycine-2C'4. This general approach led to the data reported
here on the incorporation of glycine into the acid
soluble nucleotides of rat liver and Flexner-Jobling
carcinoma and into the acid-soluble nucleotides
found in the nuclear and cytoplasmic fractions of
the Flexner-Jobling
carcinoma.
violet absorbing compounds in this extract have been recently
described in a series of papers from this institute. Busch et aL.
(4) have described the technic and apparatus which allows
Dowex-1-formate
columns to be eluted with a continuously in
creasing concentration
of eluant. The application of this meth
ad to the separation of acid-solublenucleotideshas been fully
described by Huribert et at. (15) and Schmitz et at. (22). The
method involves chromatography
on Dowex-1-formate
col
umns with two types of eluants, each of which gives different
elution patterns for known nucleotides. This permits charac
terization of a number of the acid-soluble substances. The acid
soluble components on the Dowex-1-formate columns are first
eluted with formic acid followed by formic acid containing am
monium formate. A representative
chromatogram
of this type
(designated as Type I) for acid-soluble extracts of rat liver and
Flexner-Jobling
carcinoma has been published by Schmitz et
vi. (23). In later experiments, further purification of peaks from
these chromatograms
was achieved by rechromatography
of
each peak on the Type II system of Hurlbert et al. (15), in
which ammonium formate at pH 5 was used as the eluant. This
AND METHODS
This research was supported in part by a grant from the
American Cancer Society on the recommendation of the Com
further
resolution
of the peaks.
The adenine nucleotides were characterized further by
1 Obtained
from
2 Obtained
from
S Abbreviations
a grant from the Wisconsin Section of the American Cancer
Society, and by the Alexander and Margaret Stewart Fund.
Holtzman
Tracerlab,
Rat
Co.,
Inc.,
on allocation
Madison,
Wis.
from
the
U.S.
used:
AMP,
adenosine-5-phosphate;
ADP,
adenosine diphosphate;
ATP, adenosine triphosphate;
GMP,
guanosine-5-phosphate;
GDP, guanosine diphosphate;
GTP,
guanosine
triphosphate;
IMP, inosine-5-phosphate;
RNA,
ribonucleic acid; DNA, deoxyribonucleic
acid; DPN, diphos
phopyridinenucleotide;
PCA, perchloric
acid.
t Preliminary Reports of this work have been published in
Fed. Proc., 12:199, 1953; Fed Proc., 13:202, 1954.
August
system permitted
Atomic Energy Commission.
mittee on Growth of the National Research Council, in part by
for publication
intra
Chromatographyoft/is acid-SOLUbLe
extracts.—Themethods of
chromatography and the identification of many of the ultra
The experiments were carried out on 140—160gm. female rats' bearing multiple Flexner-Jobling
Received
was injected
a liquid-air-cooledmortar as described by LePage
out for extended
The nuclear ribonucleic acid (RNA) appeared to
@
2.5 Mg. of glycine-2-C'4
counts/mm/mg)2
peritoneally into each rat. The rats were sacrificed
by freezing in liquid air. The tumors and livers
were dissected in the frozen state and powdered in
time periods after the administration
of orotic
acid-6-C'4, ITurlbert and Potter (13, 14) demon
strated a striking shift of the radioactivity
from
the acid-soluble pyrimidine nucleotides to the cor
responding nucleic acid pyrimidine nucleotides.
MATERIALS
transplants.
(20,000,000
16, 1954.
93
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
94
Cancer Research
chromatography
on paper with the n-propanol and ammonia
solvent systems described by Eggleston and Hems (9) for the
separation of AMP, ADP, and AlT. Guanine nucleotides were
identified by their location on the Dowex chromatograms
and
by isolation and characterization of the guanine released from
them by acid hydrolysis. Inosinic acid was characterized by
comparison of its chromatographic
behavior on the Dowex-1formate columns with an authentic sample and by isolation and
characterization of the hypoxanthine released from it by acid
hour to obtain the purine bases, and the bases were purified
as describedabove.
Preparation of nuclei.—For these experiments the rats were
sacrificed by decapitation,
and the tissues were rapidly re
moved to iced isotonic sucrose. Since nuclei were being cx
amined for the existence of acid-soluble components, special
precautions had to be taken to prevent gross contamination
by
cytoplasmic components or losses of acid-soluble components
by washing. To minimize cytoplasmic contamination without
subjecting the nuclei to excessive washing, a 20 per cent
hydrolysis. Uric acid was isolated and characterized
by oem
parison with a known sample on ion exchange resins and paper
homogenate
chromatograms. DPN was characterized by its chromato
graphic properties on Dowex-1 and by the reaction with KCN
tissue, and it was immediately diluted to 5 per cent with iso
tonic sucrose and was centrifuged at 600 X g, as described by
described by Colowick et al. (6).
Confirmation of the identity of all nucleotides in these ex
periments was provided by the isolation on paper chromato
grams of the purine base from an acid hydrolysate of each nu
cleotide. The bases were identified by spectral analyses, chro
matographic
properties on Dowex-SO columns, and by their
Schneider and Hogeboom (25). This high dilution insured a
minimum of mechanical contamination
with non-nuclear, non
R, values on the i.voamyl-disodiumphosphate system of Carter
(5), and the tert-butanol-HCIsystem of Smith and Markham
(29).
Preparation of purinesfor radioactivity analyses.'—Since the
nucleotide samples could be shown to contain, in some cases,
radioactivity
which was not associated with the purine base,
radioactivity
measurements
were made on the purine bases
obtained from the hydrolysis of the nucleotide in 1 N HC1. To
eliminate the sources of contamination,
the hydrolysate
was
diluted to 0.5 M HC1 and run onto a 6 X 12-mm. Dowex-SO
column (H@ form). The uric acid was not retained on the col
umn but was recovered from the eluant by chromatography on
paper
with
the
isoamyl-disodium
phosphate
system
(5). The
column was then washed with water followed by a 4-ml. per
tion of 1 M HC1. This removed any pyrimidine nucleotides
which remained on the column. A second washing with 4 ml.
of 1 M HC1 removed the hypoxanthine. Subsequent washings
with 2 and 3 as HC1 removed guanine, and adenine was re
moved with 4-0 at HC1 (30). Analyses were made with a Beck
man DU spectrophotometer
on the successive fractions as they
were eluted, in order to characterize each base as it was re
moved. The fractions were then taken to dryness in a vacuum
desiccator.
They were taken up in a small volume of water and
were spotted on Whatman No. 1 paper sheets 18 X 22.5
inches. Descending chromatography was carried out on either
one of the two solvent systems previously
described (5, 29).
The papers were air-dried, and the purines were detected under
an ultraviolet
light. The spots were cut out as circular discs,
30 mm. in diameter, and these were counted directly in inter
nal gas flow counters to a statistical error of less than 10 per
cent, except where otherwise noted. Corrections were made for
the self-absorption of the paper. The purmnesfrom the papers
were eluted in 1.0 N HC1, and the quantity of base present was
determined by measurement of ultraviolet absorption in the
Beckman
spectrophotometer.
Isolation of nucleic acids.—Theadenine and guanine of the
particulate
in isotonic sucrose was prepared
acid-soluble
nudeotides.
from the minced
The nuclei
were
then
ira
mediately extracted with 0.4 M PCA, as was the remaining
“cytoplasmic―
fraction. These extracts were chromatographed
on Dowex-1-formate columns as previously described for cx
tracts of the whole homogenate.
RESULTS
Our experiments were undertaken with the as
sumption that the most significant time after in
jection of the isotope at which to investigate the
acid-soluble extract of tissues for nucleic acid pre
cursors would be that period before nucleic acids
become significantly labeled. The experiments of
Tyner et a!. (30) indicated that activity from the
glycine begins to appear in nucleic acids about an
hour after isotope administration.
With this infor
mation as a guide, several preliminary experiments
were carried out at intervals between 20 and 120
minutes,
with different
dose levels of glycine-2-C'4.
AMP, ADP, and ATP were isolated from both
liver and tumor in these experiments, and the spe
cific activities of the three adenine nucleotides in
each tissue were very similar at each time interval
studied. The demonstration
of glycine incorpora
tion into nucleotides within 20 minutes after injec
tion suggested that studies could be carried out at
shorter time intervals. This was desirable, since,
even at 20 minutes, there was rapid equilibration
of isotope among the adenine nucleotides. Accord
ingly, an experiment was carried out with shorter
time intervals, and the data are presented
in
Table 1.
The higher specificactivity of inosinicacid rela
total nucleic acids of the tissues were extracted with hot PCA,
tive to adenylic
a modification of the procedure of Schneider (24). The bases
(Chart 2) is of note, since it indicates that this
were purified on Dowex-50 columns and isolated from paper
chromatograms
as described by LePage (16).
In experiments where DNA and RNA of the nucleus and
nucleotide
was not primarily
amination
of AMP.
cytoplasm were isolated, the hot sodium chloride extraction of
Barnes
and Schoenheimer
(1), as modified
by Hurlbert
and
Potter (13), was used to extract the mixed nucleic acids. The
DNA and RNA of nuclei were separated as described by Tyner
et al. (30). The nucleic acids were hydrolyzed
4We acknowledge
the assistance
in 1 N HC1 for 1
of Mrs.Dorothy
McManus
and Mrs. Edith Wallestad for the C'4 determinations.
acid in both
liver
formed
and tumor
by the de
The specific activity of the total adenine of the
extract is somewhat lower than that of the AMP.
This is explained by subsequent discoveries of un
identified adenine nucleotides in the extract which
do not become labeled at these time intervals.
The rapid equilibration
of isotope between the
three adenosine phosphates is apparent in both
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
EDMONDS
AND LEPAGE—Incorporation
liver and tumor, although in liver the AMP has a
higher specific activity at 10 minutes. This rapid
equilibration
even in 10 minutes indicated that it
would be desirable to work at shorter times, al
though only in tumor would it be feasible, since any
lower level of incorporation
in liver nucleotides
would not permit an accurate measurement
for
radioactivity.
Experiments were done in which the
rats were sacrificed 8 minutes after the injection of
glycine. The results of one such experiment are
shown in Table 2. Very little glycine was incor
TABLE
1
JOBLING CARCINOMA
10 Mniurzs
@
@
@
@
@
@
Liver
50 MnruTza
cpm/pM
Tumor
Tumor
Liver
18
82
32
62
1200
---
@—@-----@--@______
—SPECIFICACTIVITY
@T0TAL
TUMOR
ACTIVITY
8MINUTES
200
I2OO11LI1J1L@LL
fl
I
C,
-U
TUMOR
I0M@NUTES200
I—@@
S,
0
Tumor
0
0
17
7
21
13
36
Acid-soluble
62
405
154
1 ,360
242
3,780
75
662
257
1,500
378
4,050
55
595
136
357
adenine*
AMP-S
ADP
135
408
of an aliquot
TABLE
SPECIFIC ACTiVITIES
1,150
3,530
297
245
of the acid-soluble
2
fl
OF RAT
JOBLING
CARCINOMA
,.ooIj_I_@I_j
LIVER
AND
I__ __
Cl,
IOMINUTEO50
—
fl—
@_
AMP ADP AlP
GMP GOP GTP IMP
DPN
CHART 1.—Specific activities and total activities of acid
soluble nucleotides of rat liver and Flexner-Jobling rat carci
noma 8 and 10 minutes after the administration
of glycine-2ClS.
iNJECTION
OF
TABLE
NUCLEOTIDE
GLY
CONTENT
FLEXNER-JOBLING
<100
< 50
<25
245
Lzvza
<81
395
TuMoR
Lzvza
No. de
termi
nations
nations
ILM1gm tissue
3
3
3
0.45 (o.ss—o.64)@
0.54 (0.43—0.63)
0.47 (0.43—0.56)
GDP
2
2
0.082(0.080—0.084)
0.109(0.106—0.113)
GTP
IMP
4
4
0.190(0.069—0.210)
0.145(0.084—0.200)
AMP
ADP
Al?
estimates
of these
because
of the dif
OF LIVER AND
CARCINOMA
No. de
termi.
<5
<5
porated into nucleotides in this experiment, but it
is reported here to show that inosinic acid is la
beled before any appreciable labeling of adenine
nucleotides occurred in either liver or tumor. By
pooling tissues from several rats it became pos
sible to isolate guanine nucleotides, uric acid, and
DPN. In one experiment the animals were sacri
ficed 8 minutes after injection, and in the other 10
minutes after the glycine injection. The results of
the experiments presented in Chart 1 permit a
comparison of the total amount of isotope incor
porated into each nucleotide, as well as their spe
cific radioactivities.
To obtain data for the total
radioactivity
in each nucleotide, it was necessary
to determine the total amounts of these nucleo
Precise
possible
3
AFTER
cpm/pM
of tissue.
were not
-@ -
200@@
FLEXNER
8 MINUTES
TUMOR
Al?
ii
LIVER
CINE-2-C'4
A@W
ADP
@@1
-I
3,240
5,160
extract
fl
OF ACID-SOLUBLE Nu
CLEOTIDES
INTRAPERITONEAL
400
257 1,540 324 3,180
trace 475
854
* From an acid-hydrolysate
tides/gm
quantities
95
eluted from the Dowex-1-formate.
These results
are presented in Table 3. In the 8-minute experi
@8O0
Liver
Purines
nucleotide
in liver and tumor from spectrophoto
metric data obtained
in examination
of the bands
40 Muiurr.s
Nucleic acids:
Adenine
Guanine
Al?
iMP-S
into
ficulty involved in obtaining complete separa
tion of all the individual components
of the
extract without incurring losses in handling and
due to degradation.
however, approximate
val
ues were obtained for the concentration
of each
8oO@
RATE OF iNCoRPORATION OF GLYCINE-2-C'4 INTO PURINE
NUCLEOTIDES OF RAT LIVER AND FLEXNER
@
@
of Glycine
GMP
gzM/gm tissue
2
2
2
2
0.72([email protected])*
1.11(1.10—1.12
0.57(0.56—0.58
0.17(0.11—0.24
1
0.11
2
0.19(0.12—0.27)
* Range.
ment there was significant labeling of the adenine
nucleotides of tumor but not of liver. Even at 8
minutes there was rapid equilibration
of the iso
tope among the three nucleotides. The discrepancy
between this experiment
and that reported in
Table 2 is unexplained, but it indicated that fur
ther reduction of the time interval would not per
mit reproducible results. In this experiment the
guanine nucleotides had a specific activity about
2@ times that of the adenine nucleotides. They
also had higher
The results
specific
activities
of the 10-minute
than
the IMP@
experiment
are
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
Cancer Research
96
also shown on Chart 1. Again, the specific activi
ties of the guanine nucleotides of tumor were more
than twice those of the adenine nucleotides, but
here they had about the same specific activity as
IMP. As expected, the specific activity of IMP
exceeded that of the adenine nucleotides in both
experiments. It should be noted that in all these
experiments
the total count in each compound
tended to be of the same order. This may not be
immediately apparent in Chart 1, unless it is noted
that the scale for total counts is only one-fourth
that of the scale for specific activity.
It is of interest that the adenine nucleotides of
liver became labeled before there was any signifi
cant labeling of the guanine nucleotides, in con
trast to the findings in the tumor.
TABLE 4
NUCLEARCOMPONENTS
Per cent
total
Units*
units in
homoge
of nu
cleotide nate
extract of
of wholehomogenate
Perchioricacidextract
Dry
weight
(mg.)
Units/mg
dry wt
145
0.137
81
0.1i6
166
19.8
12
of unwashed nuclei
Perchioricacidextractof
sucrose washed nuclei
9.4
tance for the success of such a demonstration. Also,
the exclusion of any appreciable amounts of cyto
plasmic nucleotides would be necessary. This lat
ter condition is normally met by several washings
and reprecipitations
of the nuclei—a procedure
likely to result in the loss of acid-soluble nuclear
components. The desirability of minimizing wash
ing led to a study of the effects of washing on the
quantities of ultraviolet absorbing material found
in the PCA extract of the nuclear preparations
of
Flexner-Jobling
carcinoma.
The data are shown
in
Table 4. The amounts of ultraviolet absorbing ma
EFFECTS OF SuCRosE WASHING ON
Perchioricacid
standard methods for isolating nuclei were modi
fled (see “Methods―)to obtain conditions which
would be most favorable for demonstrating
them
as separate entities. The preservation
of intact
nuclear membranes would be of primary impor
5.7
* A unit is defined as the product of the total volume
and optical density
at 560 m@
Both uric acid and DPN were isolated in these
experiments. The uric acid had a very low specific
activity and could thus be assumed to be a degra
dation product. DPN was not measurably labeled.
Two other unidentified adenine nucleotides, which
represent a sizable fraction of the adenine pool,
were isolated from these extracts. The location of
one of these adenine compounds on the formic acid
chromatogram
indicates that it is an adenosine
polyphosphate
described by Hurlbert et al. and
tentatively designated as Ad-X (15). The other is
associated with the IMP peak. Neither of them
contained any radioactivity.
This could account
for the lower specific activity of the total adenine
as compared with the AMP as commented
on
earlier.
Intracellular di@tribidion of acid-soluble nucleo
tides.—Since the intracellular distribution of acid
soluble nucleotides has not been fully explored, it
was of interest to know whether nuclei contained
acid-soluble nucleotides, and if so whether they
are chemically or metabolically different from the
acid-soluble nucleotides of the cytoplasm. Since it
was probable that such nucleotides would com
prise a relatively small proportion of the total free
nucleotides of the cell, certain procedures in the
terial are recorded in arbitrary units as the prod
uct of the volume of the solution and its optical
density at 260 mp. The data show that unwashed
nuclei contain about 12 per cent of the total units
of material absorbing ultraviolet light at 260 mjs.
A single sucrose wash of these nuclei resulted in al
most a 50 per cent loss of this material, but this
loss was accompanied by a nearly parallel reduc
tion in dry weight of the nuclear fraction as seen
in column 3. This loss of nuclear material could
also be observed in the reduction of the packed nu
clear volume of the washed nuclei. It was conclud
ed that these losses of ultraviolet absorbing mate
rial by washing were due mainly to the losses of
nuclear material rather than to the removal of
cytoplasmic
contaminants.
It
therefore
seemed
justifiable to conclude that the major fraction of
the ultraviolet absorbing material extracted from
the nuclei was probably present in the nuclei and
was not merely cytoplasmic contamination.
It is
highly improbable that cytoplasmic
contamina
tion can account for as much as 12 per cent of the
total units of material absorbing ultraviolet light
at 260 m&from a homogenate of this dilution. This
would be particularly
true of Flexner-Jobling
tit
mors, which have relatively
few mitochondria
(31), the component that would be expected to be
the major source of cytoplasmic contamination.
The nucleotide composition of the acid-soluble
extract of nuclei resembled that of cytoplasm.
ITowever,
the
extracts
plasm showed extensive
triphosphates
normally
of both
nuclei
and
cyto
degradation of the di- and
present in the extracts of
the whole tissues frozen in liquid air. This was to
be expected, since enzymatic degradation would
be likely to occur during the time interval required
for the separation of the nuclei from the homoge
nate. Ribose analyses of the major peaks in the
nuclear extract indicated that the nucleotides were
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
EDMONDS
AND LEPAQE—Incorporation
mainly of the ribose type, although the methods of
analysis would not be sufficiently sensitive to de
tect small deviations in the molar ratios of purmne
base to ribose.
With these procedures established,
the incor
poration of glycine into the nuclear and cytoplas
mic fractions was studied at several time intervals.
The data are summarized in TableS. Only at the
8-minute interval was there any marked difference
among the comparable adenine nucleotide frac
of Glydne
into
97
Purines
tions. At that time the AMP of the nucleus was
more highly labeled than that of the cytoplasm,
while the opposite was true for the ADP of the two
fractions. Since almost identical data were ob
tained in two separate experiments, these differ
ences appear to be real. Interpretation
of these dif
ferences is made difficult by the fact that these
substances
do not represent
the nucleotides
as they
exist in the tissues, since there has been extensive
dilution of the AMP by ADP and ATP during the
TABLE 5
In Vise INCORPORATIONOF GLYCINE-2-C'4 INTO ACID-SOLUBLENUCLEOTIDES
OF FLEXNER-JOBLING CARCINOMA
5 MINUTEs
Nuclei Cytoplasm
(cpm/pM)
Acid-soluble nucleotides:
AMP
ADP
8 Mnwvzs
Nuclei
Cytoplasm
(cpm/pM)
15 MINUTES
Nuclei
80 Muiuvzs
Cytoplasm
Nuclei
Cytoplasm
(cpm/@M)
(cpm/pM)
89714862848457905,1806,1001062256
1,120
GMP
1,020488
920
7,200
2,7204,600
8,00016
4,500
1,330700
2,450836
16,000
17,600
7,8004,600
Nudeic acids:
Cytoplasmic
RNA
Adenine
Guanine
Nuclear RNA
8100
268
Adenine
43
Guanine
57
470
1,100
Trace
5
190
DNA
Adenine
Guanine
isolation
procedure.
--- NUCLEUS
15.0
similarities
in the
time
study
S,
study
‘p
0@
— 9.0
‘C
@
a.
specific
activities
of the synthesis
nucleotides,
@
indicate
that
of the corn
parable nucleotides of the two fractions at these
time periods. The data also serve as an additional
:
12.1
The data merely
the nucleotides of these two fractions are metabo
lized in a different manner. Chart 3 emphasizes the
CYTOPLAS
@
50
which
on AMP
and IMP
Chromatographic
IMP-S
AMP-S
:
IMP-S
0
indicated
that
of the purine
confirms
the
shown
analyses
in Chart
time
2.
of the two fractions
the acid-soluble
nuclei and cytoplasmic
mono
previous
nucleotides
fractions
of the
are chemically
similar.
6.0
As was expected
@@/,1I,lf
glycine
AMP-S
A significant,
glycine
3.1
./_
10
into
than
although
DNA
that
lower,
confirms
the
of the cytoplasm.
incorporation
of
observations
of
LePage and Heidelberger (18) that DNA labeling
can be readily demonstrated
in liver and tumor
with glycine as a precursor.
LIVER
20
(31), the nuclear RNA took up
more rapidly
3O@ 40
0
20
30
40
MINUTES
CaAwra 2and 3.—(Left) Specific activity
DISCUSSION
of AMP and IMP
of rat liver and Flexner-Jobling rat carcinoma at various
times after the administration
of glycine-2-C'4. (Right) Specific
activities of add-soluble nudeotides ofnuclear and cytoplasmic
fractions of Flexner-Jobling
rat carcinoma at various times
after the administration
of glycine-2-C'4.
The rapid incorporation of glycine into acid
soluble nucleotides relative to their nucleic acid
counterparts
in both rat liver and tumor is in ac
cord with the experiments previously cited (7, 16,
21). The rapid equilibration of C'4 between AMP,
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
Cancer Research
98
ADP, and ATP and similarly with corresponding
guanine nucleotides emphasizes the necessity for
a single pathway but that they arise from a corn
mon precursor at some early stage of nucleotide
working
synthesis,
after which each nucleotide
is synthe
sized along an independent
pathway.
Such a pro
at short
precursor
time
intervals
in investigating
after
these
giving
metabolic
the
rela
tionships.
Although the data appearing in Charts 1, 2, and
3 do not permit positive conclusions to be drawn
on the interrelationships
among the purmne nucleo
tides of the acid-soluble
eliminate
extract,
some possibilities
they do appear
to
which might have been
considered.
The formation
of IMP from AMP ap
pears to be excluded by the finding that the IMP
had a higher specific activity than the AMP in all
experiments
(Charts 2 and 3) and that it became
labeled at an earlier time than any of the adenine
nucleotides
in both
liver and tumor
the other hand, an examination
activity
found
in this
The
92). On
of the total radio
nucleotide,
taken
rate of change of its radioactivity,
cate that IMP is a direct precursor
nucleotides.
(Table
radioactivity
with
the
does not mdi
of the adenine
appearing
in the
adenine nucleotide pool greatly exceeds that of the
IMP pool before the IMP has reached its maxi
mum specific activity (Chart 1). From these data
it appears that IMP can be synthesized independ
ently of AMP. This rapid and independent forma
tion of IMP from glycine in vivo is in accord with
the in vifro studies of Greenberg (11) and Buchan
an and co-workers (26, 27) on the formation of
IMP in pigeon liver extracts. Our data indicate
that the reaction is also of significance in mam
malian tissues.
Since
adenine-C'4
can be readily
nucleic acid guanine
converted
to
(3), one might expect acid
soluble AMP to be a precursor
of the guanine nu
cleotides
of the acid-soluble
extract.
Our data do
not support such an assumption, since in tumor
the guanine nucleotides have a higher specific ac
tivity than the adenine nucleotides (Charts 1 and
3). Furthermore,
guanine nucleotides do not ap
pear to be obligatory intermediates
in the synthe
sis of adenine
nucleotides,
ute experiment
tides
to be labeled
Guanine
since in liver the 8-min
(Chart
1) shows adenine
before
nucleotides
guanine
are apparently
from inosinic acid, since
ments with tumor (Chart
nucleo
nucleotides.
not formed
rect
to exist for the next 30 minutes.
fail to provide evidence for any di
interrelationship
ship of the nucleic
among
the
three
purine
mononucleotides
found
in the acid-soluble
ex
tracts, and in several cases specific interconversions
appear to be ruled out. It is probable that the
three purine nucleotides are not metabolized along
acid
and
acid-soluble
tides. If the nucleic acid nucleotides
sized from acid-soluble
nucleotides,
nucleo
are synthe
it might
be ex
pected that the nucleic acid guanine would show a
higher specific activity
than the nucleic acid
adenine. This was the result found.
The data in Table 3 indicate that acid-soluble
nucleotides are not confined to the cytoplasm but
are present in nuclei in amounts comparable to
that fraction of the cell occupied by the nucleus.
The presence of soluble nucleotides in nuclei might
be expected, since nuclei contain enzymes capable
of synthesizing nucleotides. The synthesis of DPN
from ATP and nicotinamide mononucleotide
oc
curs in nuclei (12), and it has recently been report
ed that nuclei can carry out the synthesis of UTP
from UDP-glucose-1-phosphate
and inorganic
pyrophosphate
(20, 29).
An interpretation
of the results of the experi
ments in which labeled acid-soluble nucleotides
were obtained from nuclei and cytoplasm is made
difficult by the degradation
of the more highly
phosphorylated
nucleotides, which occurs during
the isolation of nuclei. The data indicate that nu
clear and cytoplasmic nucleotides are metabolical
ly distinct but chemically similar. Acid-soluble nu
cleotides have been reported to be synthesized by
the supernatant fraction of the cell in the absence
of any particulates or nuclei (8). The explanation
of the higher specific activity of the nuclear AMP
is not clear in the light
in the 8-minute
experi
1) they have higher spe
cific activities and a higher total radioactivity than
the IMP. Chart 3 clearly shows that this relation
ship continues
These data
posal could explain the specific activity versus
time curves depicted in Charts 2 and 3, in which
incorporation of glycine into the three nucleotides
appears to proceed independently.
It is interesting to note in Table 1 that the spe
cific activity of the nucleic acid guanine exceeds
that of the adenine at the time points studied.
Parallel differences exist in the specific activities of
the corresponding acid-soluble nucleotides at these
same times, which again suggests an interrelation
of this finding.
SUMMARY
A short-term
time study has been made of the
in vivo incorporation of glycine-2-C14 into the acid
soluble purine nucleotides and into the nucleic
acids of rat liver and Flexner-Jobling
carcinoma.
The acid-soluble nucleotides
were more highly
labeled than the corresponding nucleotides of the
nucleic acids in both tissues. The tumor incorpo
rated
much
more C'4 into nucleotides
than
did the
liver at all times studied.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
EDMONDS AND LEPAGFr-4ncorporation
Groups of acid-soluble
been isolated from rat
carcinoma at very short
jection of glycine-2-C14.
a higher specific activity
in both
liver
purmne nucleotides have
liver and Flexner-,Jobling
time intervals after the in
IMP consistently showed
than AMP, ADP, or ATP
and tumor.
GMP,
GDP,
and
various
interrelationships
among
13. HUELBERT,R. B., and POTTER,V. R. A Survey of the
Metabolism
of Orotic Acid in the Rat. J. Biol. Chem., 195:
257—70,
1952.
14.
. Nucleotide Metabolism. I. The Conversion of
Orotic Acid-6-C'4
1—21,
1954.
to
Uridine
Nucleotides.
Ibid.,
209:
15. HURLBERT,R. B. ; SCHMITZ,H.; BRuaw, A. ; and Porrsm,
V. R. Nucleotide Metabolism. II. Chromatographic Sepa
ration of Acid-Soluble
23—39,1954.
Nucleotides.
J. Biol. Chem.,
209:
16. L1tPAor@,G. A. In Vitro Incorporation of Glycine-2-C'4
into Purines and Proteins.
acid-soluble
purmne nucleotides have been discussed.
Acid-soluble nucleotides have been extracted
from the nuclear and cytoplasmic fractions of
Flexner-Jobling
carcinoma. Gradient elution chro
matography of the acid-soluble extracts of the two
99
into Purines
Studies. VI. The Synthesis of DPN by Liver Cell Nuclei.
J.Biol.Chem., 197:611—20,
1952.
GTP
were more highly labeled than the corresponding
adenine nucleotides in tumor. GMP showed a high
er specific activity than IMP in tumor. The total
radioactivity
in the individual nucleotides tended
to be of the same magnitude. Using these data,
of Glycine
Cancer Research,
13:178—85,
1953.
17.
. In: Methods in Medical Research, Vol. 1. Chicago:
Year Book Publishers, Inc., 1948.
18. LEPAGE,G. A., and HEIDELBERGER,C. Incorporation of
Glycine-2-C'4 into Proteins and Nucleic Acids of the Rat.
J.Biol.Chem , 188:593—602,
1951.
tamed in each fraction. Incorporation of glycine-2C'4 into corresponding nucleotides of each fraction
19. M@tmtic, D. H. Concerning the Metabolism of Adenine
by the Rat. Biochim. et Biophys. Acta. 13:282—87,1954.
20. Mzu@s, G. T.; ONDARzA,R.; and Swm, E. E. B. The
Uridyl Transferase of Liver. Biochim et Biophys. Acta,
showed
21. Sc@ut@o,E., and Kawi@st, H. M. Nucleic Acid Synthesis
fractions
indicate
that
similar
nucleotides
some small but reproducible
are con
differences
at
early time intervals only. At later times the spe
cific activities of the corresponding nucleotides in
each fraction tended to be similar.
REFERENCES
1. BARNES,F. W., JR., and SCROENHEIMER,
R. On the Bio
logical Syiithesis of Purines and Pyrimidines.
J. Biol.
Chem., 151:123—39,
1943.
2. BENNETT, E. L. Incorporation of Adenine into Nucleotides
and Nucleic Acids of C57 Mice. Biochim. et Biophys. Acta,
11:487—496, 1953.
3. BROWN,G. B.; Rou., P. M.; Pim@m, A. A.; and CAvA
LIERI, L. F. The
Utilizations
Synthesis and as a Precursor
172:469—84,
1948.
of Adenine
of Guanine.
for Nucleic
Acid
J. Biol. Chem.,
4. Buscn, H. ; HunuranT, R. B.; and Po@rrsm,V. R. Anion
Exchange Chromatography
of Acids of the Citric Acid
Cycle. J. Biol. Chem., 196:717—27, 1952.
5. CARTER, C. E. Paper Chromatography of Purine and
Pyrimidine Derivatives of Yeast Ribonucleic Acid. J. Am.
Chem. Soc., 72:1466—71, 1950.
6. Cowwicx, S. P.; K@pi@&sr,
N. 0.; and Ciom, M. M. The
Reactions
of Pyridine
Nudeotides
with
Cyanide
and Its
Analytical Use. J. Biol. Chem., 191:447—59,1951.
7. EDMONDS, M.; DELLUVA, A. M.; and WILSON, D. W. The
Metabolism of Purines and Pyrimidines by Growing Yeast.
J. Biol. Chem., 197:251—59,
1952.
8. EDMONDS,M. P., and LEPAGE, G. A. Incorporation of Gly
cine-2-C'4 into Acid-Soluble Nucleotide Purines. Fed.
Proc., 18:202, 1954.
9. EGGLESTON,
L. V., and HEMS,R. The Separation of Adeno
sine Phosphates by Paper Chromatography and the Equi
librium Constant of the Myokinase System. Biochem. J.,
62:156—60,1952.
10. GouwAsssm, E. Formation of Adenine Nucleotides from
Adenine by Pigeon Liver Homogenates.
Nature, 171:126,
1953.
11. G @smnmto,G. R. De Novo Synthesis of Hypoxanthine
via Inosine-5-Phosphate
and Inosine. J. Biol. Chem., 190:
611—31,
1951.
12. HoonaooM, G. H., and SCHNEIDER,W. C. Cytochemical
14:159—60,
1954.
in Developing Sea Urchin Embryos. Pubbl. Staz. Zoo!.
Napoli,24:188,1953.
22. SCHMITZ,H.; HUELBERT, R. B.; and P&rrsm, V. R. Nu
c!eotide Metabolism. ifi. Mono-, Di-, and Triphosphates
of
Cytidine, Guanosine, and Uridine. J. Bio!. Chem., 209:
41—54,
1954.
23. SCHMITZ,H.; Porrsm,
V. R.; Hurn@nrmT,R. B.; and
WErra, D. M. Alternative Pathways of Glucose Metabo
lism. II. Nucleotides from the Acid-soluble Fractions of
Normal and Tumor Tissues and Studies on Nucleic Acid
Synthesis in Tumors. Cancer Research, 14:66—74, 1954.
24. SCHNEIDER, W. C. Phosphorus
sues. I. Extraction
Compounds
and Estimation
in Animal
of Desoxypentose
Tis
Nu
cleic Acid and Pentose Nucleic Acid. J. Biol. Chem., 161:
549—65,
1952.
25. SCHNEIDER, W. C., and HOGEBOOM, G. H. Intracellular
Distribution
of Enzymes. V. Further Studies on the Dis
tribution of Cytochrome
C in Rat Liver Homogenates.
J. Biol. Chem., 183:123—28,1950.
26. ScHuia@, M. P., and Buciwra.]@r,J. M. Biosynthesis of
the Purines. II. Metabolism of 4-Amino-5-imidazole Car
boxamide in Pigeon Liver. J. Biol. Chem., 196:513—21,
1952.
27. Scaui@,
M. P.; So@z, J. C.; and Bucna@w@, J. M.
Biosynthesis of the Purines. I. Hypoxanthine
Formation in
Pigeon Liver Homogenates and Extracts. J. Biol. Chem.,
196:499—512, 1952.
28. SailTE, E. E. B., and Mxu@s, G. T. Uridine Nucleotide
Compounds of Liver. Biochim et Biophys. Acta, 13:386—
400, 1954.
29. SanTE, J. D., and M@&itsii@a*,
R. Chromatographic Studies
on Nucleic
Acids.
II. The Quantitative
nucleic Acids. Biochem.
Analysis
of Ribo
J., 46:509—13, 1950.
so. TYNER,E. P.; HEIDELBERGER,
C.; and LEPAGE,G. A. In
Vivo Studies
on Incorporation
teins and Nucleic Acid Purines.
of Glycine-2-C'4
Cancer Research,
into
Pro
12:158-
64, 1952.
31. TYNER,E. P.; HEIDELBERGER,
C.; and LEPAGE,G. A. In
tracellular Distribution
of Radioactivity
in Nucleic Acids,
Nucleotides,
and Proteins Following Sinmitaneous
Ad
ministrations
of @32
and Glycine-2-C'4. Cancer Research,
13:186—203, 1953.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1955 American Association for Cancer Research.
The Incorporation of Glycine-2-C14 into Acid-soluble Nucleotide
Purines
Mary P. Edmonds and G. A. LePage
Cancer Res 1955;15:93-99.
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