Download Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff

[CANCER
RESEARCH
33, 2265-2272,
October
1973]
Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff
Ascites Rat Tumor Cells in Culture1
Martin Schaffer, Robert B. Huribert, and Antonio Orengo
The Department of Biochemistry, The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025
SUMMARY
The utilization of labeled guanosine for the biosynthesis
of RNA and DNA has been studied in cells cultured from
the Novikoff ascites tumor of the rat. Guanosine contrib
uted primarily to guanine moieties of RNA and DNA,
whereas labeled adenosine contributed to both adenine and
guanine moieties. The labeled ribose moiety of uniformly
labeled guanosine-14C did not enter a pool of ribose
phosphate intermediates, judging from lack of contribution
to adenine, uracil, and cytosine nucleosides in RNA and
DNA.
The group of enzymatic activities that catalyze the
conversion of guanine and guanosine to guanosine triphosphate (namely, guanosine kinase, purine nucleoside phosphorylase, purine nucleoside monophosphate kinase, nu
cleoside diphosphate kinase, and purine nucleoside triphosphate phosphatase) have been prepared from an extract of
Novikoff ascites cells in a single procedure by the use of
diethylaminoethyl cellulose. Gel permeation chromatography on Sephadex G-150 was used to resolve these enzymes
sufficiently to permit determination of their individual
activities, substrate specificities, molecular weight, and
other characteristics. New rapid assays were developed for
purine nucleoside kinase and phosphorylase, utilizing la
beled nucleosides with chromatography on diethylamino
ethyl paper. These techniques were designed to be useful for
the measurement of the individual enzymes of the guanosine
salvage pathway in studies of nucleotide metabolism and
therapeutic effects.
Practical methods for culture in suspension of Novikoff
ascites tumor cells and for determination of the rates of
incorporation of guanosine (and other nucleic acid precur
sors) into RNA and DNA are described. RNA and DNA
are extracted with hot 2.5 M potassium acetate and sepa
rated by use of alkali in a form convenient for resolution of
individual nucleotides and bases by electrophoresis and
chromatography.
INTRODUCTION
Although nucleic acid derivatives are not required in the
diet, the utilization of bases and nucleosides may have
'This work has been supported by N IH Research Grant ÇA-10407to
A. O. Development of the methods for cell culture and the "hot potassium
acetate" procedures for extraction and separation of nucleic acids was
supported by The American Cancer Society Research Grant P-146 to R. B.
H. Additional support was provided by Grants G-460 and G-447 from
the Robert A. Welch Foundation.
Received December 20, 1972; accepted June 8, 1973.
OCTOBER
considerable significance in the adult metazoan in which
large amounts of nucleic acids are degraded hourly in the
normal processes of destruction and renewal of cells. The
life-span of neutrophils is estimated at 70 to 80 hr (8), and
that of lymphocytes is estimated at 24 hr (7, 26). Erythrocytes are replaced at a rate of 0.83% every day (2), and 60 to
70% of the lining epithelium of the intestine is shed daily
(11). rRNA and mRNA are also degraded and are presum
ably utilized intracellularly. The possibility that these
salvage processes may be regulated or coordinated with
synthesis de novo of nucleotides has not received the proper
attention.
In this communication we report on the utilization of
guanosine for biosynthesis of nucleic acids in Novikoff
ascites rat tumor cells. These cells in tissue culture suspen
sion readily utilize 14C-labeled guanosine and adenosine for
nucleic acid biosynthesis. Here, we describe both the pattern
of utilization of the nucleosides by living tumor cells and the
partial isolation from these cells of a group of enzymes
responsible for the conversion of guanosine to GTP. It may
be useful that these enzymatic activities can be partially
purified simultaneously.
MATERIALS
AND METHODS
All the 14C-labeled nucleosides and nucleotides were
purchased from Schwarz/Mann, Orangeburg, N. Y., or
from Amersham/Searle Corp., Arlington Heights, 111.All
the other nucleosides and nucleotides used were products of
P-L Biochemicals, Milwaukee, Wis. Ribose 1-phosphate
was purchased from Sigma Chemical Co., St. Louis, Mo.
The Novikoff ascites tumor was originally supplied by Dr.
Alex B. Novikoff. Whatman DEAE-cellulose DE 52 was
purchased from H. Reeve Angel and Co., Inc., Clifton, N.
J. Amino acids and vitamins were purchased from Sigma;
bovine and calf serum was from Grand Island Biological
Co., Grand Island, N. Y., Pluronic acid F68 was from
Wyandotte Chemical Co., Wyandotte, Mich.; streptomycin
sulfate was from Charles Pfizer and Co., Inc., New York,
N. Y.; and neomycin was from the Upjohn Co., Kalamazoo, Mich.
;
The Novikoff ascites cells were transplanted and grown
for 5 to 6 days in the peritoneal cavity of young female
Holtzman Sprague-Dawley rats (120 to 150 g).
Cell Culture. For establishment of a suspension culture of
tumor cells in vitro, rats displaying modest amounts of
ascitic fluid were selected since tumor cells derived from
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2265
M. Schaffer, R. B. Huribert, and A. Orengo
large ascites volumes had a higher initial mortality when set
up in suspension culture. A rat was anesthetized and the
abdominal area thoroughly cleaned with 70% ethanol.
Using a sterile disposable 10-ml syringe and 20-gauge
needle, 4 to 5 ml of ascitic fluid were removed and diluted
with an equal amount of culture medium previously warmed
at 37°using sterile conditions. The cells were dispersed
using a gentle rocking motion and centrifuged at 60 xg for
about 5 to 6 min. The supernatant, which should contain
most of the red cells, was carefully decanted. The sedimented cells were then dispersed using the same rocking
motion in 5 ml of fresh medium at 37°.
One-tenth ml of the cell suspension was diluted to 10 ml
with culture medium and counted in an hemacytometer.
Once the number of cells in the suspension was determined,
aliquots containing 1.9 x 10' cells were transferred to
culture bottles and culture medium warmed at 37°was
added to a total volume of 25 ml. Sterile technqiues were
used at all times. The bottles were capped well and placed in
the roller drum at 37°;then they were incubated for 24 hr.
The culture medium was prepared by mixing in order the
following ingredients: a dry mixture of salts, 100 ml of a
modified McCoy's amino acid (4, 15) 10 x stock solution,
20 ml of McCoy's vitamin 50 x stock solution (15), 10 ml of
(neutral point of phenol red); 1.0 ml was withdrawn and the
bottles were returned to the roller drum. The aliquot was
then diluted with medium and counted as described above.
The desired cell number after the start of the culture was
5 x IO5cells/ml of medium and this applied as long as the
cell line was continued. Consequently, if the cell suspen
sions were grown to a density of more than 5 x IO5cells/
ml, appropriate aliquots were withdrawn and discarded or
cultured separately. The remainder was centrifuged for 5
min at 60 x g to remove the old medium and to replace it
with 25 ml of fresh, warmed medium. At the end of any
48-hr period if the cell count had not doubled the suspension
was discarded. On all subsequent feedings, the same proce
dure was followed to keep the cell count at ca. 5 x IO5
cells/ml. The cells should have a rounded appearance with a
sharp cell membrane. Cells that were very large and
granular, developing swelling or vacuoles, were regarded as
unlikely to survive and were not counted when taking a cell
count. Cells were not subjected to heavy mechanical or
thermal shock. They were not left for more than 24 hr
without changing the medium. When NaHCO3 was added,
the bottle was swirled to prevent a sudden localized massive
pH change. The bottles were not shaken but merely tilted or
rocked back and forth.
an antibiotic mixture, 50 ml of bovine serum, and 50 ml of
For the incorporation studies described here only cultures
fetal calf serum. The solution was diluted to 1 liter with that doubled their cell number for 3 consecutive days were
used.2
double glass-distilled water and sterilized by filtration
Extraction and Initial Purification of Guanosine-metabothrough a Seitz filter which had been autoclaved using a size
6 filter sheet (Republic Seitz Filter Co., Newark, N. J.). It lizing Enzymes. For studies on the purification of the
enzymatic activities, 20 to 50 tumor-bearing rats were
was stored at 04.
decapitated and the ascitic fluid was collected and diluted
The dry mixture of salts was composed of: lactalbumin
hydrolysate, 5.0 g; Pluronic F68, 1.0 g; NaCl, 8.0 g; KC1, 1:2 with 0.25 Msucrose : 1 m.MMgCl2 and then cooled. The
0.4 g; MgSO4-7H2O, 0.2 g; Na2HPO4-2H2O, 0.060 g; material was maintained at 2-4° throughout the entire
KH2PO4, 0.060 g; glucose, 3.0 g; glutamine, 0.219 g; and procedure. The fluid was then centrifuged at 200 x g, and
the tumor cells were collected as a sediment. This sediment
NaHCO3, 2.0 g.
The modified 10 x McCoy's amino acid mixture con
was repeatedly suspended in fresh sucrose solution and
tained the following amino acids (mg/liter): i.-tryptophan,
centrifuged at 200 x g to remove most of the erythrocytes.
31; i.-phenylalanine, 165; L-tyrosine, 181; L-arginine-HCl,
Finally, the cells were packed by centrifugation at 1000 x g
421; i.-histidine-HCl-H2O, 209; L-lysine-HCl, 365; L-cys- to estimate their volume and then suspended in 0.01 M
tine, 315; i.-methionine, 149; i.-isoleucine, 393; L-leucine, Tris-Cl:0.25 M sucrose (pH 7.7) at a cell to buffer ratio of
393; t.-valine, 176; i.-threonine, 179; i.-asparagine, 450; 1:4.
The suspension was homogenized in an Emanuel-Chaikglycine, 75; L-serine, 263; L-alanine, 134; L-proline, 173;
L-aspartic acid, 199; i.-glutamic acid, 221. It was stored off orifice-type homogenizer (Microchemical Specialties
Co., Berkeley, Calif.) (5). In order to break most of the
frozen.
The antibiotic mixture contained (g/liter): phenol red cells, it was necessary to pass the suspension through the
(sodium salt), 0.5; streptomycin sulfate, 5; and neomycin, 2. homogenizer twice. The homogenate was centrifuged at
20,000 x g for 20 min in a refrigerated International Model
It was stored frozen.
The complete medium should not be used after a 3-month B20 centrifuge. The supernatant was collected and cen
period; the vitamin stock solution should be discarded after trifuged again for 2 hr at 147,000 x g in a refrigerated
International Model B35 centrifuge. The sediment was
6 months.
The cell cultures were grown as suspensions in 60- x
150-mm centrifuge bottles with flat bottoms and a 38-mm
2These culture conditions were developed by Dr. C. Vaughan, with the
black plastic screw cap (Pyrex brand glass, serial 1261). The advice of Dr. M. Sheek. independently from those described tor long-term
bottles were coated with silicone and baked overnight before culture of the Novikoff ascites tumor by Morse and Potter (16). The
medium described here is more complete than that of Morse and Potter
use.
in direct comparison in our laboratory, gave consistently more
Subsequent Feeding of Cell Suspensions. At the end of the and,
favorable results in establishment of primary cultures when it was not de
24-hr period of incubation, following sterile procedures, sired to maintain cultures continually. We have found it useful in routine
NaHCO3 (7.5% solution, w/v) was added dropwise until the preparation of Novikoff tumor RNA highly labeled with ribonucleosides
color of the medium changed from yellow to orange-red or with phosphate-32?.
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GuarÃ-osmeAnabolism in Novikoff Ascites Cells
discarded and the supernatant was called Fraction 1.
Protein concentration was determined by the method of
Lowry et al. (13) and the concentration was then adjusted to
5.0 mg/ml with 0.02 MTris-Cl (pH 7.5). For each 1000 ml
of Fraction 1, 600 ml of DEAE-cellulose suspension in 0.01
MTris-Cl (pH 8.0): 10% glycerol (50:50, v/v)3 were added
slowly with continuous stirring. After 2 hr of gentle stirring
the suspension was centrifuged and the supernatant was
discarded. The sediment was then washed with 1000 ml of
H2O adjusted to make the solution pH 8.0 with NaOH.
After centrifugation the supernatant was discarded, and
the sediment was washed again with 400 ml of 0.075 M
KC1:0.01 M Tris-Cl: 10% glycerol (pH 8.0). The superna
tant was discarded.
The sediment was then eluted by 2 hr of gentle stirring in
500 ml of 0.3 M potassium phosphate buffer (pH 6.5). The
DEAE-cellulose was removed by centrifugation, and the
volume of supernatant was reduced at least 6 times using
Centriflo ultrafiltration membrane (American Scientific
Systems Division, Lexington, Mass.). This was called
Fraction 2.
Gel Permeation Chromatography. Fraction 2 was cen
trifuged briefly to remove any precipitate. Of the resulting
solution, 20 ml containing approximately 200 mg of protein
were applied in 0.3 M potassium phosphate buffer (pH 6.5)
on a Sephadex G-150 column (3 x 52 cm). The flow rate
was 15 ml/hr and fractions of 4 ml were collected. The
proteins were eluted with 0.01 M Tris (pH 7.7) and the
fractions were read at 280 nM to follow the protein elution.
The enzymatic activities were determined with the radioac
tive assays described below.
Assays of Enzymes. All enzymatic activities were deter
mined by radiochemical assays. All assays were done at 37°
and initial velocities were measured (no more than 10%
conversion allowed). In this range, the measured activities
were proportional to enzyme concentration. The products
and substrates were separated either by chromatography on
DEAE-paper (Whatman DE 81) or by high-voltage electrophoresis on Whatman No. 3MM paper.
Nucleoside Kinases Assay. A new radiochemical assay
was used which measures the conversion of labeled ñucleoside to nucleotide by Chromatographie separation of the
reactants and products on DEAE-paper. The standard assay
mixture (final volume of 60 ¿tl)contained 5 Amólesof
Tris-Cl buffer (pH 7.4), 420 nmoles of MgCl2, 300 nmoles
of ATP, 100 nmoles of nucleoside labeled with 14Cin the
purine or pyrimidine ring (1 /Ã-Ci//¿mole),and varying
amounts of enzyme. After 1 hr of incubation, 25-^1 aliquots of the reaction mixture were pipetted in duplicate
as spots 5 cm from 1 end of DEAE paper strips 2.9 x 14cm
(previously spotted with 0.1 /¿moleof unlabeled nucleoside
as carrier), dried, and subjected to descending chromatog
raphy. The solvents used were isobutyric acid:H2O:NH3
(65.7:34:2:0.1) for the adenosine and guanosine and 80%
ethanol for the uridine and cytidine. Whereas the nucleosides move with the solvent, the nucleotides remain at the
3The volume ratio of DEAE-cellulose and solution was determined
after centrifugation of an aliquot for 10 min at 1000 x g.
origin. An average 1% of the counts remains on the origin at
0 incubation time. This value may vary with the purity of
the commercial preparations of the radioactive nucleosides
used. The nucleotide spots were located by UV illumination,
cut out, and counted in a Nuclear-Chicago low-background
flow counter. The products were shown to be GMP and
ADP by electrophoretic assays. The entire procedure takes
2 hr.
Purine Nucleoside Phosphorylase Assay. The standard
assay mixture (final volume of 60 ^1)contained 10 Amólesof
Tris-Cl (pH 7.4), 50 nmoles of guanosine-U-14C,4 100
nmoles of sodium phosphate buffer (pH 7.4), and suitable
amounts of enzyme. The incubation time was 15 min. The
Chromatographie system was the same as that used for the
nucleoside kinase assay. Since the guanosine used is labeled
both in the guanine and ribose, this allowed measurement of
the ribose 1-phosphate produced. Whereas the nucleoside
and base moved with the solvent, the ribose 1-phosphate
produced by the phosphorylase remained at the origin.
Carrier guanosine and 5'-GMP were also added as de
scribed for nucleoside kinase. 5'-GMP was added as a UV
marker for the position of the ribose 1-phosphate with no
UV absorbance. The spots remaining at the origin were cut
out and counted in a Nuclear-Chicago low-background flow
counter.
In pilot experiments the location of ribose 1-phosphate at
the origin was established by chromatography of ribose
1-phosphate and spraying with aniline : acetic :orthophosphoric acid reagent (6:200:20) (22).
Assays for Nucleoside Monophosphate and Nucleoside
Diphosphate Kinases and for Purine Nucleoside
Triphosphatase. The standard assay mixture (final volume,
50 n\) contained 5 Amólesof Tris-Cl buffer (pH 7.7), 300
nmoles of ATP, 700 nmoles of MgCl2, and 50 nmoles of
GMP-8-14C or GDP-U-14C or GTP-8-l4C plus a suitable
amount of enzyme. The reactions were incubated at 37°for
10 min and stopped by cooling the mixture to near 0°.In a
volume of 30 /tl, 100 nmoles each of guanosine, GMP,
GDP, and GTP were added. Aliquots of 5 or 10 ^1 were
spotted
13 cm
fromv(56".5'
the cathodic
of ainsheet
of Whatman
No. 3MM
•¿'paper
x '29:5endcm)
duplicate.
The
compounds were separated by paper electrophoresis on 0.05
Mcitrate buffer (pH 3.5) on a flat-plate apparatus. The field
strength of the system was 47 V/cm and the temperature
was kept between 5 and 7°.Each spot was localized by UV
illumination, cut out, and counted in a Packard liquid
scintillation counter. Assays for the other nucleoside kinases
were conducted by substitution of appropriately labeled
nucleotides for the guanosine nucleotides. The RI-TP, i.e.,
relative migration with respect to UTP for the nucleotides,
can be listed as follows: UMP, 0.548, UDP, 0.882; GMP,
0.420; GDP, 0.741; GTP, 0.821; AMP, 0.252; ADP, 0.583;
ATP, 0.735.
One unit of enzyme was defined as the amount catalyzing
the conversion of l ^mole of substrate in 60 min under the
condition of the standard assay for all of the previously
' U refers to the uniformly labeled compound.
OCTOBER 1973
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2267
M. Schaffer,
R. B. Huribert,
and A. Orengo
described assays. The counting efficiencies were 72% for the
scintillation counter and 9.5% for the flow counter.
Hot Potassium Acetate Procedures for Extraction and
Analysis of RNA and DNA. Ten ml of a 5% trichloroacetic
acid solution were added to 0.5 ml of packed ascites cells.
After centrifugation the sediment was washed 2 or 3 times
with 10 ml of 5% trichloroacetic acid and then with 10 ml of
95% ethanol, rinsing the entire inner surface of tube and
breaking up the precipitate. After centrifugation, the super
natant was discarded and the tube was drained well.
Three ml of cold 2.5 M potassium acetate and 1 small
drop of concentrated phenol red solution were added to the
sediment. While still cold, the suspension was neutralized to
pH 7.4 to 8.0 with 1.0 and 0.1 M KOH. The suspension was
stirred well and allowed to stand for 10 min at room
temperature to ensure equilibration and diffusion into
particles. The pH was adjusted if necessary; if too alkaline,
RNA will subsequently be lost; if too acid, both RNA and
DNA will be lost. The tube was capped, heated with
occasional stirring for 30 min in a boiling water bath, and
chilled in ice. After centrifugation, the supernatant was
removed by means of a disposable pipet and filtered through
a small plug of glass wool to remove particles. The pellet
was reextracted in the same way with 1 ml of 2.5 M
potassium acetate, with heating for 5 to 10 min. The 2
supernatants were combined and 2 to 2.5 volumes of
absolute ethanol were added. The suspension was stirred
well and set in a deep freeze for several hr. The flocculent
precipitate of potassium nucleates was centrifuged down
and washed with 7 ml of cold 70 to 80% ethanol. After
centrifugation the supernatant was removed as completely
as possible with a disposable pipet. Recovery of DNA is
about 90% and of RNA is about 80%, based on comparison
with assays by the Schneider method (Ref. 19, cf. Ref. 9).
The DNA loss is primarily due to the mechanical manipula
tions and it is suspected that some tRNA is lost due to
slight degradation and solubility in the ethanol precipita
tion.5
In order to separate RNA from DNA, 1 ml of 0.2 N
NaOH (carbonate free) was added to the sediment, and the
solution was incubated at 37°for 16 to 18 hr in a capped
tube. At the end of the incubation the tube was cooled and
0.1 ml of 4.4 N perchloric acid was added. The DNA
precipitate was centrifuged down in the cold, and the RNA
hydrolysate was removed by disposable pipet and trans
ferred to another small tube.
The DNA precipitate was washed without delay with
several ml of cold 0.2 N perchloric acid. The supernatant
was discarded and the tube was drained well. The DNA was
redissolved in 1.0 ml of 0.2 N NaOH and reprecipitated with
5The extraction with 2.5 M potassium acetate is preferable to the
extraction with 10%NaCI solution previously described (18) because of the
more convenient buffering capacity of potassium acetate during neutraliza
tion and its greater solubility in ethanol to reduce salt concentration in the
electrophoretic analysis. The procedure is highly reproducible and conven
ient for isotope determinations on components of RNA and DNA in small
tissue samples, when relatively specific precursors of nucleic acids are used.
Where lipids and phospholipids are a problem, washing of the precipitated
tissue or of the potassium nucleates with ethanol:ether and ethanol is
necessary.
2268
cold perchloric acid as before to ensure removal of the last
traces of ribonucleotides. The pellet was then dissolved in
0.5 ml of 0.5 M NaCI, 1 drop of phenol red was added, and
the solution was neutralized. Ethanol, 1.5 volumes, was
added, and the mixture was set in the freezer for several hr.
The precipitate was centrifuged and washed by suspension
and centrifugation in 1 ml of ethanol. The precipitate was
dried and then heated for 2 hr at 175°in 91% formic acid.
The hydrolysis was carried out in a sealed pyrex tube. The
hydrolysate was evaporated under reduced pressure to
dryness and redissolved in 100 to 300 n\ of l N HC1.
Aliquots ( 10 to 20 ¿il)
taken for Chromatographie separation
of the bases were applied on Whatman No. 1 paper and
subjected to descending chromatography using the solvent
system of Wyatt (25). In no case was radioactive uracil
detected indicating that the DNA was free of RNA.
The RNA hydrolysate [2'(3')-nucleotides] was prepared
for paper electrophoresis as follows. The solution was
adjusted to pH 3.5 with KOH, chilled near freezing to
maximize precipitation of KC1O4, and centrifuged. Ali
quots (20 /il) of the supernatant were applied as a line on a
sheet of Whatman No. 3MM filter paper, and paper
electrophoresis was carried out in 0.05 Mcitrate buffer (pH
3.5). The field strength of system was 47 V/cm and the
temperature was kept between 5 and 7°.The nucleotides
were separated in the following order going from the
negative to the positive pole: cytosine, adenine, guanine, and
uracil.
The paper was dried and the bases or the nucleotides were
located by UV and cut out. The paper spots were immersed
in a scintillator solution (4 g of PPO and 50 mg of POPOP
per liter of toluene) and counted in a liquid scintillation
counter.
RESULTS
Table 1 shows that 14C-labeled nucleosides are readily
incorporated into cultured Novikoff rat ascites cells. The
patterns of incorporation into nucleic acids are distinctly
different for adenosine and guanosine. When guanosine-814C is used as precursor of RNA and DNA, the label is
found predominantly in guanine moieties, whereas adenosine-8-14C contributed strongly to both guanine and adenine
moieties. From the experiments with guanosine-U-14C
labeled in both guanine and ribose, it appears that the ribose
of the guanosine did not contribute significantly to a
common pool of ribose 1-phosphate or 5-phosphoribosylpyrophosphate for other nucleotide biosynthesis. One would
expect more radioactivity in the pyrimidine nucleotides if
the labeled ribose 1-phosphate derived from the action of
guanosine phosphorylase were available. The ready utiliza
tion of adenosine for the synthesis of both AMP and GMP
suggest that adenosine may be utilized through conversion
of its deamination product, inosine, to inosinic acid as well
as interconversion of AMP and GMP via inosinic acid at
the nucleotide level. These results are in line with those
obtained by Williams and LePage (23, 24). These authors
studied the in vivo and in vitro incorporation of preformed
purines into nucleotides and polynucleotides using incuba
tion periods varying from 5 to 60 min.
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Guanosine Anabolism in Novikoff Ascites Cells
Table 1
Incorporation of "C-labeled guanosine and adenosine into RNA and DNA of Novikoff tumor cells maintained in suspension cell cultures
l4C-Labeled nucleosides (0.5 ^Ci/¿Ã-mole),
0.5 ml, were added to 100 ml of cell culture suspension (5 x 10s cells/ml). The bottles were then capped
and placed in a roller drum at 37°and incubated with continuous motion for 12 or 24 hr. At the end of the incubation period the cells were harvested by
centrifugation and washed twice with medium, and nucleic acids were extracted and analyzed as described in the text. Part of the cell culture suspension
was maintained without radioactive nucleoside and used as a control to follow cell division. RNA was hydrolyzed to 2'(3')-nucleotides and DNA to the
bases guanine, adenine, cytosine, and thymine, which were separated by electrophoresis and chromatography as described in the text.
RNA2'(3')-CMP00.50.70.82'(3')-AMP2.52.61.353.12'(3')-GMP95.795.297.445.82'(3')-UMP1.81.70.60.3G89.791.
of labeled
ofDNAA4.14.37.769.8C2.42.62.53.6T3.82.02.50.7
labeled
-(^moles/5
added
x10'
cells)1.861.861.801.36%
Guanosine-U-"C12
hr24
hrGuanosine-8-"C24
hrAdenosine-8-14C24
hrAmount
A similar pattern of incorporation has been reported for
the reticulocyte (3) and the erythrocyte of the rabbit (14).
Again, guanine and guanosine-8-'"C was utilized extensively
for GTP synthesis but to a very limited extent for the
synthesis of ATP.
In order to study and characterize the enzymatic machin
ery responsible for these in vivo findings, it was necessary to
assay for the guanosine-anabolizing enzymes. Fraction 2, as
described, was the only fraction of the cell extract that
metabolizes guanosine. It was observed to convert guano
sine to a number of metabolites, and therefore it was not
possible to assay specifically for the individual enzymes
involved.
The resolution of these enzymes was attempted and gel
permeation chromatography on Sephadex G-150 was found
to yield a sufficient resolution of the enzymatic activities
responsible for the incorporation of guanosine into guano
sine nucleotides. The enzymes were not in all cases com
pletely separated from each other but in every case a specific
assay was possible. For example, in the case of purine
nucleoside phosphorylase and nucleoside diphosphate kinase the enzymes were not resolved but the specificity of the
assays distinguish them. The former is assayed with labeled
guanosine and P,, the latter with labeled GDP and ATP.
The cluster of enzymatic activities included guanosine
kinase, purine nucleoside phosphorylase, purine nucleoside
monophosphate kinase, nucleoside diphosphate kinase, and
purine nucleoside triphosphate phosphatase (Charts 1 and
2). Chart 3 shows the apparent molecular weights of these
enzymatic activities as obtained by the gel permeation
chromatography method to be: purine nucleoside monophosphate kinase, 1.9 x IO4,purine nucleoside triphosphate
phosphatase, 3.2 x 10*; nucleoside diphosphate kinase, 6.2
x IO4and guanosine phosphorylase, 6.8 x 10*.No apparent
molecular weight could be determined for guanosine kinase
since the enzymatic activity was eluted with the void
volume. It was not possible to determine amounts of
individual enzymes in the initial extract or Fraction 2
because substrates and products would be acted upon by the
other enzymes in the sequence. Hence, we cannot present
information on recoveries.
Substrate Specificity Studies. The center tube of the peak
labeled guanosine kinase was shown to convert guanosine
only to GMP when the products were analyzed by the
electrophoretic analysis. This fraction was tested for activity
as adenosine kinase with no conversion to nucleotides.
Uridine kinase activity was present in the guanosine kinase
preparation here described but the possibility that I enzyme
was responsible for both activities was excluded on the
finding that (a) highly purified uridine kinase from Novikoff
tumor cells (17) was tested with this assay and did not
exhibit guanosine kinase activity (The lower limit of
detectability for guanosine kinase in the conditions of our
assay is 0.01 unit/ml. A 90-fold purified preparation of
uridine kinase (32 units/ml) shows no guanosine kinase
activity.) and (¿>)
unlabeled uridine (120 nmoles) does not
compete with 14C-labeled guanosine for the kinase.
The nucleoside phosphorylase appears to be specific for
guanosine since uridine and adenosine could not be used as
substrates.
Table 2 shows that the peak labeled NMK in Chart 2
catalyzes the phosphorylation of GMP to GDP but does not
phosphorylate UMP. AMP is phosphorylated to ADP to a
minor extent. This may be due to contamination by
adenylate kinase or may be a property of the guanylate
kinase itself; we have not explored this point further.
However, Entner and Gonzales (6) reported that the
reproductive tract of Ascaris lumbricoides contains a high
level of AMP and GMP kinase. They also presented
evidence of a partial separation of the guanylate from the
adenylate kinase. More recently, Shimono and Sugino (21)
have reported the purification of an enzyme that catalyzes
transphosphorylation between ATP, GMP, and dGMP.
The enzyme has been purified 1000-fold from extracts of
calf thymus and exhibits a strict specificity for these
nucleotides, which contain guanine as the base component.
Nucleoside diphosphate kinase is present in very high
levels in Novikoff ascites rat tumor cells. We find little or no
substrate specificity in this activity (Table 3) as is the case
with the enzyme prepared from other sources (1, 10, 18).
The nucleoside triphosphatase activity observed had no
activity on pyrimidine nucleotides (CTP, UTP, or TTP) and
OCTOBER 1973
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2269
M. Schaffer, R. B. Huribert, and A. Orengo
30
40
50
60
FRACTION
70
80
90
100
110
NUMBER
Chart I. Elution of guanosine (G/î)kinase andguanosine phosphorylase
from a Sephadex G-150 column. An aliquot öl"Fraction 2 was applied to a
Sephadex
G-150 column (3 x 52 cm) equilibrated
7.7. The same buffer
Fractions of 4 ml
enzymatic activities.
radiochemical assays
with 0.01 MTris-CI, pH
was used to elute the enzymes at a rate of 15 ml/hr.
were collected, read at 280 nm, and assayed for
The standard conditions described in the text for
were used. Va, void volume.
converted GTP to GDP 1.75 times faster than ATP to
ADP. Considerable amounts of nucleoside triphosphatase
activity were removed by the pH 8.0 water wash.
metazoan to any significant extent, but rather they are
derived primarily by degradation of nucleic acids. They may
be so reinserted into nucleic acids as nucleotides resulting
from the actions of nucleotide pyrophosphorylases, nucleo
side phosphorylases, and kinases. We believe that salvage
pathways must have greater significance in the adult
metazoan, which degrades large amounts of nucleic acids,
than is generally considered. Tumor metabolism seems to
depend to a great extent on salvage mechanisms, and these
mechanisms must be considered in design and comprehen
sion of chemotherapeutic approaches involving biosynthesis
of nucleic acids. Rapid and convenient procedures for
determining incorporation in vivo of guanine precursors
into RNA and DNA, in conjunction with isolation, partial
resolution, and measurement of activities of the enzymes
involved in the incorporation, may be of interest in studies
of the effects of certain chemotherapeutic agents on the
biosynthesis of RNA-guanine in sensitive and resistant
strains of tumor. We expect that different tissues and
tumors will utilize purines with different metabolic patterns;
therefore, these patterns should be specifically determined
for each system.
It is also conceivable that the coordinated and normal
functioning of salvage pathways may exercise reciprocal
controls on the de novo synthesis of purines. The LeschNyhan (12) syndrome seems to substantiate such an infer
ence. The syndrome is an X-linked neurological disorder
consisting of mental retardation, choreoathetoses, cerebral
palsy, and a typical compulsive biting of fingers and lips
DISCUSSION
In this paper we have described convenient methodology
for culture of Novikoff ascites tumor cells and a practical
and reliable method for determination of the rates of
incorporation of guanosine (and other nucleosides or nucleic
acid precursors) into RNA and DNA. These methods have
a wide range of applicability in addition to the specific
studies here. In addition, the group of enzymatic activities
that catalyze the conversion of guanosine to GTP have been
prepared from an extract of these same cells in a single
procedure by use of DEAE-cellulose, and methods are
presented for sufficient resolution of these enzymes by gel
permeation chromatography to permit study of their activi
ties and characteristics.
Although the enzymatic reactions of guanosine metabo
lism here described have been known for some time to occur
in certain types of cells and/or bacteria, the results of these
studies interest us inasmuch as they offer an accessible
system in order to study (a) details of specificities of
substrates and phospho-donors in assimilation of guanine in
a single cell type and (b) the biosynthesis of GTP from
guanine or guanosine by a multienzyme system recon
structed in vitro. As a result, interrelated phenomena such
as competition for substrates, rate-limiting steps, and
regulatory mechanisms of allosteric nature could be investi
gated in a controllable model system accessible by relatively
simple analytical techniques.
Bases and nucleosides are not synthesized de novo in the
2270
130
NOK
120 100 '-
90 80 r_ 70 E
to
t 60 3
2.0 I
Õ 50>PNl
*
40 -
30
I.OK
20 -
CO
tr
o
IO -
20
30
40
50
60
FRACTION
70
80
NUMBER
90
100
110
Chart 2. Elution of purine nucleoside monophosphate
kinase (NMK),
nucleoside diphosphokinase
(NDK), and GTPase from a Sephadex G-150
column. The conditions are identical to the ones described in Chart 1.
CANCER RESEARCH
VOL. 33
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Guanosine Anabolism in Novikoff Ascites Cells
Table 3
Substrate specificity of nucleoside diphosphokinase from Novikoff
rat tumor asciles cells
The reaction mixture contained 5 //moles of Tris-Cl buffer (pH 7.7),
0.7 /jmole of MgCl2, 0.3 /imole of ATP, the labeled nucleoside triphosphates in the concentrations listed below, and 2.45 /ig of enzyme (Frac
tion 63 from the Sephadex column) in a total volume of 60 ¡i\.The reac
tion mixtures were incubated for IOmin at 37°.At the end of the incuba
tion time, 75 nmoles each of the appropriate mono-, di-, and nucleoside
triphosphates in a total volume of 20 /il were added to the reaction mix
ture which was immediately frozen in a bath of Dry Ice and acetone. Ali
quots of 10 //I were spotted on Whatman No. 3MM paper and the com
pounds were separated by paper electrophoresis in 0.05 M citrate buffer.
pH 7.0. Each spot was localized by illumination with UV, cut out, and
counted in a Packard scintillation counter.
2.5CYTOCHROME
NMK 19x10
CHYMOTRYPSINOGEN'
i GTPose 3.2 x IO4
2.0-
OVALBUMIN
NDK 62
ALBUMIN
t IO '
> GR Fhosphorylose
(bovln«)«\6.8
x IO4
•¿X
1.5y GLOBULIN
(human)
y GLOBULIN
V
activity(cpm/nmole)436550561.61239.0Amount
ofXTP
tration(mM)0.8160.8681.2000.918Radio
inIO formed
SubstrateGDP-U-'K:ADP-U-"CUDP-U-14CCDP-2-"CConcenmin(nmoles)33.137.039.035.3
DIMER (?)
1.0-
a!
(o
A 1
00.
z z
•¿-—¿â€”¿I
—¿I
0.5
IO3
IO4
IO5
Molecular
Weight
—¿I
with consequent mutilations. An enzyme defect associated
with the syndrome is indeed a lack of hypoxanthine (guanine) phosphoribosyltransferase activity detectable in sev
Chart 3. Determination of the molecular weights of guanosine phospho- eral tissues of the affected subjects (20). Since these patients
rylase (GR phosphorylase), purine nucleoside monophosphokinase
have a marked increase in the rate of purine biosynthesis de
(NMK), nucleoside diphosphokinase (N[>K), and GTPase by filtration on novo, the deficiency of the salvage enzyme strongly indi
Sephadex G-150. Mixtures of 10 mg bovine serum albumin and I mg
cates a coordination between the 2 convergent pathways but
cytochrome c, 8 mg chymotrypsinogen A and ovalbumin, and 5 mg human
-y-globulin and I mg of cytochrome in volumes of 5 ml were filtered does not yet define the biochemical lesion responsible for
through a column of Sephadex G-150 (3 x 52 cm) equilibrated with 0.01 M the physiological signs.
IO6-
Tris-Cl. pH 7.7, in 3 separate runs. Cytochrome c was determined by
following the absorbance at 412 nm. All the other molecular markers were
determined by following the absorbance at 280 nm. In a separate run 210
mg of protein (Fraction 2) in a volume of 16 ml were filtered through the
column. The enzymatic activities were determined by the radiochemical
assays as described in the text.
Table 2
Substrate specificity of purine nucleoside monophosphokinase from
Novikoff ral tumor osciles cells
The reaction mixture contained 5 //moles of Tris-Cl buffer (pH 7.7),
0.7 //mole of MgCl2, 0.3 /mióleof ATP. and 53.8 nmoles of AMP-8"C (980 cpm/nmole), 48.0 nmoles of UMP-2- "C ( 1144 cpm/nmole). or
38.8 nmoles of GMP-8-"C (1114 cpm/nmole), and 1.9 //g of enzyme
(Fraction 82 from the Sephadex column) and was incubated in a total vol
ume of 50 /il for 20 min at 37°.
Adenosine, AMP, ADP, and ATP, 150 nmoles each, or uridine, UMP,
U DP, and UTP. or guanosine, GMP, and GTP, 150 nmoles each, in a
volume of 30 /¿I
were added to the reaction mixture which was immediately
frozen in a bath of Dry Ice and acetone. Aliquots of 10 //I were spotted on
Whatman No. 3MM paper, and the compounds were separated by paper
electrophoresis in 0.05 Mcitrate buffer, pH 3.4. Each spot was locali/ed by
illumination with UV, cut out, and counted in a Packard scintillation
counter.
Amount formed in 20 min (nmoles)
Substrate
UDP
UTP
ADP
ATP
GDP
GTP
0.0AMP-8-14CGMP-8-"C0.04.2
UMP-2- "C
2.819.7
5.7
ACKNOWLEDGMENTS
We wish to thank Dr. Caroline Vaughan for developing the optimal
conditions for cell culture, Cynthia K. Parks for assistance in establishing
the "hot potassium acetate procedure," and Frances J. Estes for excellent
technical assistance.
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CANCER RESEARCH VOL. 33
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Guanosine Anabolism for Biosynthesis of Nucleic Acids in
Novikoff Ascites Rat Tumor Cells in Culture
Martin Schaffer, Robert B. Hurlbert and Antonio Orengo
Cancer Res 1973;33:2265-2272.
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