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Incorporation of radioactivity from thymidine into mammalian glucose and glycogen
393
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97091
INCORPORATION OF RADIOACTIVITY FROM T H Y M I D I N E INTO MAMMALIAN GLUCOSE AND GLYCOGEN R. L O W R Y D O B S O N A.ND M A R Y F A I T H C O O P E R
Bio-Medical Division, Lawrence Livermore Laboratory, University o[ Cali[ornia, Livermore, Call]. 94550 (U.S.A.)
(Received July 27th, 1971)
SUMMARY I. Glycogen and DNA were isolated b y CsC1 density-gradient centrifugation from livers of mice injected with [x4C]thymidine or [l*Clthymine while in specified nutritional states. Amounts of radioactivity found in glycogen varied with feeding patterns and reached levels far exceeding those in DNA. Polysaccharide labeling occurred in normally fed animals, but was heaviest in previously fasted ones that were fed prior to injection and were therefore actively synthesizing both glucose and glycogen. Glycogen labeling was Io-fold greater after EMe-X4C3thymidine than after [2-14C]thymidine. 2. Chromatography of acid hydrolysates of the radioactive glycogen and response of the product to glucose oxidase showed all of the radioactivity to be in glucose. 3. The similarity of these findings to those we have made in the flatworm indicates that in widely separated animal species substantial amounts of radioactive label from catabolized exogenous DNA precursor molecules can be incorporated into glucose and then into glycogen. Polysaccharide thus labeled in vivo may, in a number of experimental circumstances, be mistaken for newly synthesized DNA.
INTRODUCTION Contamination of nucleic acids with polysaccharides has been recognized to occur in various isolation procedures t-6. In some cases complex-formation has been described ~,e. In addition, COUNTS AND FLAMM~ found small amounts of radioactivity associated with liver glycogen in mice injected with E14C]thymine. While warning of possible interference with estimates of DNA synthesis, they interpreted the labeling to be an artifact caused by binding of nucleoside phosphate. More recently PIK& reported incorporation of [l~C]thymidine into polysaecharlde, thought not to be glycogen, as well as into DNA in mouse embryos cultured in v#ro. We observed in other studies 8 that radioactivity from administered ~C-labeled thymidine and thymine, and uridine as well, appeared prominently in a component banding close to DNA in CsC1 density gradients of planarian tissue. This component was not present following pretreatment with e-amylase. Analysis showed it to be glycogen and the radioactivity to be in glucose. Since the planarian DNA was not Biochira. Biophys. Aeta, 254 (1971) 393-4ol
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R.L. DOBSON, M. F. COOPER
labeled, we were led to re-examine reported studies on nucleic acid synthesis in flatworms, and noted that radioactivity in polysaccharide had been mistaken for newly formed DNA and RNA. This suggested that catabolism of adminstered nucleic acid precursors followed b y incorporation of radioactive products into glucose and then glycogen might be important also in the mammal and might be the real explanation of results such as those repolted by COUNTS AND FLAMMI. This mechanism could account for the unexplained labeling kinetics they observed as well as their other findings. It could account also for the observations by DEVIK AND HALVORSEN9 of discrepancies between biochemical and autoradiographic determinations of DNA synthesis in liver and for similar findings b y others 1°. The results on mice reported here demonstrate that after administration of labeled thymidine or thymine, liver glycogen can indeed become radioactive, not by artifact but b y incorporation of newly synthesized radioactive glucose. The magnitude of this effect can be very great: glycogen can become enormously more radioactive than DNA. MATERIALS AND METHODS Female Swiss mice weighing 23-27 g were fed in three different ways. Normally fed animals had food pellets (Wayne Lab-Blox, Allied Mills, Inc.) always available. Fasting animals were deprived of food for 48 h prior to injection of tracer and not fed subsequently. Fasted-fed animals were similarly deprived, then presented with food 5-15 rain (or in one case, I h) before injection. All mice had free access to water. Sterile aqueous solutions at IOO/~C/ml of [2-14C1 thymine (18-56 mC/mmole) and of [2-1~Clthymidine (44-53 mC/mmole) and [Me-l*Clthymidine (55 mC/mmole) (New England Nuclear) were checked b y radioassay and confirmed b y thin-layer chromatography on cellulose, with three different solvent systems, to be the pyrimidine and nucleoside, respectively. The solvent systems were distilled water, n-butanol-acetic acid-water (80 : 12 : 30, by vol.) and ethyl acetate-formic acid-water (60 : 5 : 35, b y vol.). In the thymine preparations a very small second radioactive peak was seen on radio-scans of chromatograms done with the first two. Its R F w a s somewhat less than that of thymine with the second solvent system; with distilled water it was somewhat greater, and corresponded to the slight (less than 2.5 %) radio-contamination reported by the supplier. No radio-contamination was detected in the thymidine solutions. Each mouse was injected inttaperitoneally with 45/zC of either thymine or thymidine, and at various times between I and 24 h the animals were killed quickly by neck luxation and the livers immediately removed and frozen.
Separation o/ DNA and glycogen by CsCI gradient ultracentri]ugation Approximately 13o mg of tissue was removed from each liver, weighed, brought to 5o mg/ml in i % sodium lauryl sulfate (Sigma Chemical Corp.), i mM EDTA, io ml~ Tris (Trizma Base, Sigma Chemical Corp.) buffer, pH 8.o and homogenized in a Potter-Elvehjem tissue grinder. The homogenate was stirred at 2o ° for I h. Pronase (Calbiochem) was added to 5°/zg/ml, and stirring was continued for 2 h. The preparation was dialyzed overnight against sodium lauryl sulfate buffer.
Biochim. Biophys. Acta, 254 (I97I) 393-4Ol
RADIOACTIVE GLYCOGENFROM INJECTED THYMIDINE
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Into i ml of the dialyzed homogenate was dissolved 1.215 g CsC1 (Radio Tracer Grade, Harshaw Chemical Co.) to give a density of 1.69 g/ml. After a Io-min centrifugation at 9ooo×g, I ml was removed from below the surface material and layered over I ml of higher density (1.77 g/ml) CsC1 solution in a cellulose nitrate ultracentrifuge tube (No. 331226, Beckman Instruments, Inc.). For the higherdensity solution, 1.4oo g CsC1 was dissolved in I ml of i mM EDTA, io mM Tris (pH 8.0) buffer. I ml of a lower density solution (I.6o g/ml), similarly prepared but with i.OlO g CsC1, was layered over the sample. The tubes were subjected to 80 ooo ×g in a 40.3 fixed-angle rotor in a Model L2-65B Spinco ultracentrifuge for 60 h at 20 °.
Analyses o] gradient ]ractions From 28 to 35 fractions were collected by gravity from each gradient. Fractions were diluted with 0.2 ml distilled water, and absorbance was measured with a Zeiss PMQ II spectrophotometer at 260 nm for nucleic acid and at 320 nm for light scattering by glycogen (which was visible as an opalescent band in the gradient tubes). Polysaccharide was determined also by a modified diphenylamine method sensitive to hexose but not to nucleic acids 3. Radioactivity was measured on 2oo-/~1 samples in a liquid scintillation counter (Mark I, 6860, Nuclear-Chicago) at 8 ° with 15 ml of the following scintillant: 3 kg 1,4-dioxane, 300 g naphthalene (recrystallized) (both, Eastman Kodak Co.), 15 g PPO and 1.8 g dimethyl-POPOP (both, Packard Instrument Co.). Counting efficiency was 86 %. CsC1 in the gradient samples did not cause significant quenching.
Hydrolysis and analysis o[ polysaccharide Fractions comprising the polysaccharide peak of a given gradient were pooled, dialyzed against distilled water at 4 °, and lyophilized. The polysaccharide was tested for sensitivity to Qt-amylase (EC 3.2.1.1) (Worthington Biochemical Corp.) by incubation with the enzyme at 25 ° in 20 mM sodium phosphate (pH 6.9) corttaining 6 mM NaCI. The concentration of enzyme was 2/~g/ ml and that of polysaccharide, I.O mg/ml. The liberation of reducing groups was measured by reduction of 3,5-dinitrosalicylic acid. Hydrolysis for chromatographic analysis was carried out by dissolving the polysaccharide ill I M HCI to give 250/~g/ml and heating for I h at IOO°. The hydrolysate was dried over solid NaOH in a vacuum dessicator, twice washed with distilled water and redfied. Analysis for sugars was by methods modified from LEWIS AND SMITH11. Aqueous solutions of hydrolysate were applied to thin-layer cellulose chromatography plates (M2g-Polygram Cell 3oo, Brinkmann) and developed by two ascents with pyridineethyl acetate-acetic acid-water (36 : 36 : 7 : 21, by vol.) (Solvent system I.) Various sugars were chromatographed on the plates for comparison. Carbohydrate components were visualized by spraying with o.I M p-anisidine and phthalic acid in 95 % ethanol and heating. Radioactivity was located with a chromatogram radio-scanner (Model 6ooo-lo, Varian Aerograph). Radioactive material was recovered from nonsprayed spots by scraping the cellulose from the areas into distilled water. Glucose was determined with a glucose oxidase reagent (Glucostat Special, Worthington Biochemical Corp.).
Biochim. Biophys. Acta, 254 (1971) 393-4oi
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R. L. DOBSON, M. F. COOPER
Glucose was also enzymatically converted to gluconic acid using a method modified from BENTLEY AND NEUBERGER12. As a o.I % solution in IO mM sodium phosphate (pH 6.8), it was incubated at 22 ° with I/zg/ml glucose oxidase (EC 1.1.3.4) (Worthington Biochemical Corp.) and IOO/~g/ml catalase (EC 1.11.1.6) (Calbiochem) for 24 h. Products of this reaction were chromatographed on thinlayer silica gel plates (Polygram Sil N-HR, Brinkmarm). Two solvent systems were used, chloroform-methanol (60 : 40, by vol.) (Solvent system II) and butanol-acetic acid-water (5o : 25 : 25, by vol.) (Solvent system III). Radioactivity was located as before, and spots were visualized by spraying with 0. 5 % potassium permanganate in I ~][ NaOH. Comparison solutions were prepared with glucose (J. T. Baker Chemical Corp.) and gluconic acid (Sigma Chemical Co.).
RESULTS Fourteen mice were included in the feeding experiments, seven normally fed, one fasting, and six fasted-fed. The 48-h fast followed by refeeding was to ensure gluconeogenesis and stimulated glycogen synthesis at the time of tracer administration.
Glycogen labeling in normally/ed mice From E2-1iC]thymidine. Two normally fed animals were injected with E2-14C]thymidine and killed at 3 and 7 h. Gradient data from the 3-h mouse are presented in Fig. IA. The approx. 35/zg DNA present in the sample contains a total of 400 counts/min; the glycogen, amounting to 270/zg, contains a hardly detectable, estimated 4o counts/rain. Data from the 7-h animal were practically identical, except that the glycogen was not detectably radioactive. From [Me-14C]thymidine. One mouse received [Me-14Clthymidine and was killed at 2 h. The gradient pattern and DNA labeling were similar to those of the previous two animals. However, approx. IOO #g of glycogen in this case contained 500 counts/min, IO times greater than that in the glycogen of the [2-14C]thymidineinjected mouse of Fig. IA and more than was contained in the DNA. From E2-14C~thymine. Four animals were injected with E2-14C~thymine and killed at 3, 7 and 24 h. Gradient patterns were essentially the same as before, but DNA was not measurably radioactive and only the 3-h mouse showed detectable labeling of glycogen, estimated at 40 counts/rain as in the thymidine-injected mouse of Fig.iA. Thus, glycogen labeling was seen in normally fed animals only at the earlier times examined, 2 and 3 h after tracer injection. When x4C was in the 2-carbon position of the administered material, radioactivity appearing in polysaccharide was barely above the limit of detectability for the methods and sample-sizes used here. After thymidine with 14Cin the methyl group, however, it was an order of magnitude greater, and the amount of radioactivity in glycogen exceeded that in DNA.
Heavy labeling during active glucose and glycogen synthesis Of the six fasted-fed animals, three received radiothymidine and three radiothymine. They were killed between i and 6 h after injection. All six showed marked radioactivity of the polysaccharide. In Fig. IB are data from a 3-h E2-1aClthymidine-
Biochim. Biophys. Acta, 254 (1971) 393-4oi
397
RADIOACTIVE GLYCOGEN FROM INJECTED THYMIDINE
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Fig. I. CsC1 d e n s i t y g r a d i e n t s of liver h o m o g e n a t e s f r o m mice in v a r i o u s n u t r i t i o n a l s t a t e s a t t h e t i m e t h e y were injected w i t h [l~C]thymidine or [14C]thymine. Main b a n d D N A IDNAm) a n d satellite (DNA~) d e t e r m i n e d b y A , 0 . m , p o l y s a c c h a r i d e (P) d e t e c t e d b y l i g h t - s c a t t e r i n g a t 32o n m a n d m e a s u r e d as h e x o s e b y d i p h e n y l a m i n e , a n d r a d i o a c t i v i t y as c o u n t s / r a i n are s h o w n p e r t o t a l u n d i l u t e d fraction. U l t r a c e n t r i f u g a t i o n a n d fraction a n a l y s e s were as described in MATERIALS AND ~[ETHODS. S e d i m e n t a t i o n is f r o m r i g h t to left. A. N o r m a l l y fed a n i m a l , 3 h a f t e r i n j e c t i o n w i t h [2-z*C]thymidine, ]3. F a s t e d - f e d a n i m a l , 3 h a f t e r b e i n g fed a n d injected w i t h [2-14CJthym i d i n e . C. F a s t e d - f e d a n i m a l , 4 h a f t e r b e i n g fed a n d injected w i t h [Me-Z4C]thymidine (note t h a t t h e r a d i o a c t i v i t y scale h a s b e e n c o m p r e s s e d io-fold). D. F a s t i n g a n i m a l , 2 h after injection w i t h [ 2 - " C ] t h y m i n e . E. F a s t e d - f e d a n i m a l , 2 h a f t e r b e i n g fed a n d injected w i t h [2-1*C]thymine. F. F a s t e d - f e d a n i m a l , 5 h a f t e r b e i n g fed a n d 4 h a f t e r i n j e c t i o n w i t h [2-1~C]thymine.
Biochim.Biophys.Acta,254
(1971) 3 9 3 - 4 o i
398
R. L, DOBSON, M. F. COOPER
injected mouse. The prominent radioactive peak in this gradient is glycogen, not DNA. The 560 pg present in the sample contains 20 times more radioactivity than is present in the 35 Pg DNA. While there is more polysaccharide than in Fig. IA, it represents glycogen that has been newly synthesized during the 3 h of refeeding, as liver glycogen stores are depleted by a 48-h fast (cf. Fig. ID). In fig. IC are the gradient data from a fasted-fed mouse injected, in this case, with EMe-14C]thymidine and killed 4 h later. While there is less glycogen than in Fig. IB, the labeling is an order of magnitude greater. The 325/~g polysaccharide in this gradient contains 200 times more radioactivity than does the 35 pg DNA. A similarly injected mouse, whose data are not shown, was killed after only I h. Its very much smaller amount of glycogen had the same specific activity as that in Fig. IC, suggesting that after receiving thymidine labeled in the methyl group, the fasted-fed mouse continues for some hours to incorporate 14C into glycogen in about the same proportion as during the first hour after injection. In Fig. ID are data from a fasting mouse killed 2 h after injection of [2-1ac]thymine, and in Fig. IE are data from a fasted-fed animal also killed 2 h after injection with the same material. No glycogen is detectable in the fasting mouse, and no radioactive labeling is seen associated with either the DNA or glycogen. The fastedfed animal, however, while showing no detectable radioactivity in DNA, shows a markedly radioactive glycogen peak. This represents polysaccharide synthesized during the 2 h of refeeding. The total radioactivity is almost identical to that in the similarly fasted-fed, but thymidine-injected, mouse of Fig. IB. An animal like that of Fig. IE, but sampled 6 h after injection, had a glycogen peak 4 times larger, indicative of the quantity of polysaccharide synthesized during the additional 4 h. The total radioactivity in the peak, however, was the same, suggesting that incorporation of 14C into glycogen from injected I2-14C]thymine is complete by 2 h. This contrasts with the more extended incorporation of radioactivity following injection of [Me-14Clthymidine noted above. Fig. I F shows data from a fasted-fed animal given food a full hour before injection with E2-14C]thymine, and killed 4 h after injection. Diphenylamine determinations of hexose are not available, but from the A3~0nravalues the amount of glycogen appears to be slightly more than that in Fig. IE. There is less labeling however, presumably because refeeding had produced a decrease in gluconeogenesis by the time of injection. A striking thing about this gradient is the position of glycogen well within the DNA region, emphasizing that it is not always separated from DNA even by ultracentrifugation in CsC1 gradients. Identification of radioactive glucose as the cause of glycogen labeling The polysaccharide isolated from CsC1 gradients responded to treatment with ¢¢-amylase by degradation as shown by a liberation of reducing groups. Chromatography of acid hydrolysates of the radioactive polysaccharide yielded a single spot with the staining properties of carbohydrate. It showed the specific color reaction of hexose 11. As seen in Fig. 2A, this spot had the same RF as glucose, and contained all the radioactivity. The eluate of the radioactive spot was positive for glucose when tested with the highly specific glucose oxidase reagent. A further stringent test of identity was applied. The eluted hydrolysate material of an unsprayed radioactive spot was reacted with glucose oxidase under conBiochim. Biophys. Acta, 254 (1971) 393-4Ol
399
RADIOACTIVE GLYCOGEN FROM INJECTED THYMIDINE A G
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Fig. 2. Thin-layer chromatogram and radio-scan tracings showing positions of stained sample spots and of radioactivity relative to origin (O) and solvent front (SF). Sample preparation and solvent systems were as described in MATERIALS AND METHODS. A. Glucose control (G) and hydrolysate of radioactive polysaccharide (H) on cellulose with Solvent system I, and radio-scan of H lane. B. Chromatogram on silica gel with Solvent system II of glucose control (G), control reaction mixture consisting of glucose with glucose oxidase and catalase (G + E), and the reaction mixture consisting of eluted hydrolysate spot from cellulose with glucose oxidase and catalase ( H + E ) , and radio-scan of H + E lane. C. Same as 13 except t h a t Solvent system III was used.
ditions that completely convert any glucose present TM, and rechromatographed. As seen in Fig. 2B and C, the product of the control glucose-enzyme reaction (G+E) remained at the origin with Solvent system II and showed a n R F of 0.2 5 with Solvent system III (as did gluconic acid when chromatographed with these systems). The product of the hydrolysate--enzyme reaction (H+E) behaved identically. The absence of any spot at the glucose position in both G + E and H + E lanes indicates that conversion of glucose was complete. All of the radioactivity behaved identically to glucose before reaction with glucose oxidase, and identically to the product, gluconic acid, afterwards, demonstrating that the 1'C was incorporated in glucose.
DISCUSSION
These experiments show that variable amounts of radioactivity can be incorporated into mouse liver glycogen after administration of radio-thymidine or thymine. This labeling can be vastly greater than that of DNA. Polysaccharide becomes especially radioactive when the tracer is injected at a time of active synthesis of both glucose and glycogen. The significance of this to studies involving DNA synthesis derives from the readiness with which polysaccharide can contaminate and be confused with nucleic acid 1-e. That confusion may occur even when isopycnic centrifugation methods are used for isolating DNA is illustrated by the overlap of DNA and glycogen shown in Fig. IF. Some investigators4 report banding positions similar to those in Fig. IA--E; Biochim. Biophys. Aaa, 254 (197 x) 393-4oi
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R. L. DOBSON, M. F. COOPER
others s report glycogen to have precisely the same buoyant density as mouse DNA. The cause of variations in buoyant of density glycogen in CsC1 gradients is under study. How much error might radioactive glycogen introduce into estimates of DNA synthesis? Perspective on this question can be obtained from data in Fig. IC. The 320/~g of glycogen are seen to contain 9 ° ooo counts/rain, 28o counts/rain per #g. The 35/zg of DNA can be estimated to contain about 400 counts/rain, or I I counts/ rain per/zg. Now if, unbeknown to the investigator, this DNA were 35 % contaminated with glycogen (Note that M_~ARTINEZSEGOVIA e~ al. ~, using phenol extraction methods on mouse liver, reported persistent polysaccharide contamination of DNA in the ratio 0.54 : I.), then I #g of the DNA (containing I I counts/rain) would appear to contain 162 counts/rain. This would be a 14oo % error. Similarly, a io °/o contamination would produce an error of some 300 %, and a contamination as low as I o/ /o would lead to a 25 % erroi. If DNA is not significantly radioactive, as in the thymine-injected mice reported here (see Fig. IE), then any counts in glycogen mistakenly attributed to DNA could lead to even greater percentage errors. On the other hand, if similar considerations are based on data from a [2-14Clthymidine-injected mouse, as in Fig. IB, errors would be smaller b y a factor of IO than those based on Fig. IC. Thus a wide range of uncertainty m a y exist unless the investigator can be sure that this phenomenon plays no significant role in his experimental results. The liver, in regard to the effect under consideration, represents a case in which DNA synthesis is normally at a low level while glycogen metabolism is prominent. This would be expected to maximize the effect. Muscle might be somewhat similar, but the phenomenon should be less important in rapidly renewing cell systems. Studies on other tissues are in progress. That the mechanism of liver glycogen labeling is through incorporation of radioactive glucose, itself newly formed from thymine breakdown products, is apparent from the results reported here together with known features of thymidine catabolism 18, gluconeogenesis 14 and glycogen synthesis 15. The more intense and more sustained active labeling of glycogen after EMe-14Clthymidine than after E2-14C]thymidine is accounted for b y the fact that much of the a4C from the 2-carbon position is metabolized quickly to CO2 and can be lost, while that in the methyl group is metabolized to fl-aminoisobutyric acid and is more available for gluconeogenesis xe. Labeling of glycogen after EMe-14C!thymidine but not after E2-14C]thymidine has recently been reported in a protozoan 17. Our results accord with the findings of COUNTS AND FLAMM4 as regards magnitude of labeling following injection of I2-14C]thymine into normally fed animals and its decrease after the first few hours. However the m a x i m u m labeling they reported was Io-fold less than we observed in a normally fed mouse given EMe-14C]thymidine. It is likely that normal levels of gluconeogenesis and glycogen synthesis account for the relatively low-grade labeling seen in normally fed animals shortly after injection and that the decline after a few hours is due to mobilization of glucose from outer labeled tiers of glycogen molecules (c]. STETTEN AND STETTEN18).That such a decline was not observed in the fastedfed mice of these experiments is explained b y the radioactive glucose being incorporated into deeper portions of the newly synthesized glycogen molecules and therefore not so readily mobilized. Effects from natural diurnal variations in feeding and metabolic cycles are under investigation. Biochim. Biophys. Acta, 254 (1971) 393-4Ol
RADIOACTIVE GLYCOGEN FROM INJECTED THYMIDINE
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The [Me-14C]thymidine-injected mouse of Fig. IC incorporated 3 % of the administered radioactivity into liver glycogen. The [2-14C]thymidine-injected animal of Fig. I B incorporated 0. 3 %. The latter figure is about the same as that which can be derived from the data of SCHNEIDER AND GRECO 19 for total incorporation observed in various fractions of liver particulate preparations, including lipid, protein, DNA, RNA, and acid-soluble, after injection of EMe-3H]thymidine and [2-14C~thymidine. The fact that the mouse of Fig. IC incorporated IO times more than this into liver glycogen alone illustrates how greatly the labeling of non-DNA components~ glucose and glycogen in particular, can depend upon metabolic states at the time of radiothymidine administration.
ACKNOWLEDGMENT
The authors wish to thank Dr. Frederick T. Hatch and Joseph A. N[azrimas for helpful suggestions during the course of these experiments and for review of the manuscript. This work was performed under the auspices of the U.S. Atomic Energy Commission. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Atomic Energy Commission or of the University of California to the exclusion of others that may be suitable. REFERENCES I O. T. AVERY, C. M. MACLEOD AND I~. McCARTY, J . Exp. Med., 79 (1944) 137. 2 J. MARMUR, J. Mol. Biol., 3 (1961) 2o8. 3 Z. M. ~ARTINEZ SEGOVIA, F. SOKOL, I. L. CvRAVESAND W. W. ACKERMANN, Bioehim. Biophys. Acta, 95 (1965) 329. 4 W. :B. COUNTS AND W. G. FLAMM, Biochim. Biophys. Acta, 114 CI966) 628. 5 L. PIK6, A. TYLER AND J. VINOGRAD, Biol. Bull., 132 (1967) 68. 6 D. G. PRITCHARD, R. ~¢~. HALPERN AND R. A. SMITH, Biochim. Biophys. Acta, 338 (1971) 127. 7 L. PIK6, Devel. Biol., 21 (197o) 257. 8 R. L. DOBSON AND M. F. COOPER, m a n u s c r i p t in preparation. 9 F. DEVIK AND K. HALVORSEN, Nature, 197 (1963) 148. i o L. O. CHANG AND W. B. LOONEY, Cancer Res., 25 (1965) 1817. I I B. A. L E w i s AND F. SMITH in E. STAHL, Thin-Layer Chromatography, Springer-Verlag, N e w York, 1969, p. 8o 7. 12 R. BENTLEY AND A. NEUBERGER, Biochem. J., 45 (1949) 584 . 13 J" E. CLEAVER, Thymidine Metabolism and Cell Kinetics, N o r t h Holland, A m s t e r d a m , 1967, P.54 14 H. A. LARDY, The Harvey Lectures, Series 6o, Academic Press, New York, 1966, p. 261. 15 H. Cy. HERS AND H. DEWULF, Syrup. on Control o[ Glycogen Metabolism, Proc. 4th Meeting Fed. Eur. Biochem. Socs., Oslo, I967, Universitetsforlaget, Oslo, 1968, p. 65. 16 K. FINK, R. E. CLINE, R. B. HENDERSON AND R. •. FINK, J. Biol. Chem., 22i (1965) 425 . 17 P. A. LANZETTA AND J. BERECH, JR., Fed. Proc., 3° (1971) 1124. 18 M. R. STETTEN AND D. STETTEN, JR., J. Biol. Chem., 213 (1955) 723 • 19 ~V. C. SCHNEIDER AND A. E. GRECO, Bioehim. Biophys. Acta, 228 (1971) 61o.
Biochim. Biophys. Acta, 254 (1971) 393-4Ol
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97091
INCORPORATION OF RADIOACTIVITY FROM T H Y M I D I N E INTO MAMMALIAN GLUCOSE AND GLYCOGEN R. L O W R Y D O B S O N A.ND M A R Y F A I T H C O O P E R
Bio-Medical Division, Lawrence Livermore Laboratory, University o[ Cali[ornia, Livermore, Call]. 94550 (U.S.A.)
(Received July 27th, 1971)
SUMMARY I. Glycogen and DNA were isolated b y CsC1 density-gradient centrifugation from livers of mice injected with [x4C]thymidine or [l*Clthymine while in specified nutritional states. Amounts of radioactivity found in glycogen varied with feeding patterns and reached levels far exceeding those in DNA. Polysaccharide labeling occurred in normally fed animals, but was heaviest in previously fasted ones that were fed prior to injection and were therefore actively synthesizing both glucose and glycogen. Glycogen labeling was Io-fold greater after EMe-X4C3thymidine than after [2-14C]thymidine. 2. Chromatography of acid hydrolysates of the radioactive glycogen and response of the product to glucose oxidase showed all of the radioactivity to be in glucose. 3. The similarity of these findings to those we have made in the flatworm indicates that in widely separated animal species substantial amounts of radioactive label from catabolized exogenous DNA precursor molecules can be incorporated into glucose and then into glycogen. Polysaccharide thus labeled in vivo may, in a number of experimental circumstances, be mistaken for newly synthesized DNA.
INTRODUCTION Contamination of nucleic acids with polysaccharides has been recognized to occur in various isolation procedures t-6. In some cases complex-formation has been described ~,e. In addition, COUNTS AND FLAMM~ found small amounts of radioactivity associated with liver glycogen in mice injected with E14C]thymine. While warning of possible interference with estimates of DNA synthesis, they interpreted the labeling to be an artifact caused by binding of nucleoside phosphate. More recently PIK& reported incorporation of [l~C]thymidine into polysaecharlde, thought not to be glycogen, as well as into DNA in mouse embryos cultured in v#ro. We observed in other studies 8 that radioactivity from administered ~C-labeled thymidine and thymine, and uridine as well, appeared prominently in a component banding close to DNA in CsC1 density gradients of planarian tissue. This component was not present following pretreatment with e-amylase. Analysis showed it to be glycogen and the radioactivity to be in glucose. Since the planarian DNA was not Biochira. Biophys. Aeta, 254 (1971) 393-4ol
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R.L. DOBSON, M. F. COOPER
labeled, we were led to re-examine reported studies on nucleic acid synthesis in flatworms, and noted that radioactivity in polysaccharide had been mistaken for newly formed DNA and RNA. This suggested that catabolism of adminstered nucleic acid precursors followed b y incorporation of radioactive products into glucose and then glycogen might be important also in the mammal and might be the real explanation of results such as those repolted by COUNTS AND FLAMMI. This mechanism could account for the unexplained labeling kinetics they observed as well as their other findings. It could account also for the observations by DEVIK AND HALVORSEN9 of discrepancies between biochemical and autoradiographic determinations of DNA synthesis in liver and for similar findings b y others 1°. The results on mice reported here demonstrate that after administration of labeled thymidine or thymine, liver glycogen can indeed become radioactive, not by artifact but b y incorporation of newly synthesized radioactive glucose. The magnitude of this effect can be very great: glycogen can become enormously more radioactive than DNA. MATERIALS AND METHODS Female Swiss mice weighing 23-27 g were fed in three different ways. Normally fed animals had food pellets (Wayne Lab-Blox, Allied Mills, Inc.) always available. Fasting animals were deprived of food for 48 h prior to injection of tracer and not fed subsequently. Fasted-fed animals were similarly deprived, then presented with food 5-15 rain (or in one case, I h) before injection. All mice had free access to water. Sterile aqueous solutions at IOO/~C/ml of [2-14C1 thymine (18-56 mC/mmole) and of [2-1~Clthymidine (44-53 mC/mmole) and [Me-l*Clthymidine (55 mC/mmole) (New England Nuclear) were checked b y radioassay and confirmed b y thin-layer chromatography on cellulose, with three different solvent systems, to be the pyrimidine and nucleoside, respectively. The solvent systems were distilled water, n-butanol-acetic acid-water (80 : 12 : 30, by vol.) and ethyl acetate-formic acid-water (60 : 5 : 35, b y vol.). In the thymine preparations a very small second radioactive peak was seen on radio-scans of chromatograms done with the first two. Its R F w a s somewhat less than that of thymine with the second solvent system; with distilled water it was somewhat greater, and corresponded to the slight (less than 2.5 %) radio-contamination reported by the supplier. No radio-contamination was detected in the thymidine solutions. Each mouse was injected inttaperitoneally with 45/zC of either thymine or thymidine, and at various times between I and 24 h the animals were killed quickly by neck luxation and the livers immediately removed and frozen.
Separation o/ DNA and glycogen by CsCI gradient ultracentri]ugation Approximately 13o mg of tissue was removed from each liver, weighed, brought to 5o mg/ml in i % sodium lauryl sulfate (Sigma Chemical Corp.), i mM EDTA, io ml~ Tris (Trizma Base, Sigma Chemical Corp.) buffer, pH 8.o and homogenized in a Potter-Elvehjem tissue grinder. The homogenate was stirred at 2o ° for I h. Pronase (Calbiochem) was added to 5°/zg/ml, and stirring was continued for 2 h. The preparation was dialyzed overnight against sodium lauryl sulfate buffer.
Biochim. Biophys. Acta, 254 (I97I) 393-4Ol
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Into i ml of the dialyzed homogenate was dissolved 1.215 g CsC1 (Radio Tracer Grade, Harshaw Chemical Co.) to give a density of 1.69 g/ml. After a Io-min centrifugation at 9ooo×g, I ml was removed from below the surface material and layered over I ml of higher density (1.77 g/ml) CsC1 solution in a cellulose nitrate ultracentrifuge tube (No. 331226, Beckman Instruments, Inc.). For the higherdensity solution, 1.4oo g CsC1 was dissolved in I ml of i mM EDTA, io mM Tris (pH 8.0) buffer. I ml of a lower density solution (I.6o g/ml), similarly prepared but with i.OlO g CsC1, was layered over the sample. The tubes were subjected to 80 ooo ×g in a 40.3 fixed-angle rotor in a Model L2-65B Spinco ultracentrifuge for 60 h at 20 °.
Analyses o] gradient ]ractions From 28 to 35 fractions were collected by gravity from each gradient. Fractions were diluted with 0.2 ml distilled water, and absorbance was measured with a Zeiss PMQ II spectrophotometer at 260 nm for nucleic acid and at 320 nm for light scattering by glycogen (which was visible as an opalescent band in the gradient tubes). Polysaccharide was determined also by a modified diphenylamine method sensitive to hexose but not to nucleic acids 3. Radioactivity was measured on 2oo-/~1 samples in a liquid scintillation counter (Mark I, 6860, Nuclear-Chicago) at 8 ° with 15 ml of the following scintillant: 3 kg 1,4-dioxane, 300 g naphthalene (recrystallized) (both, Eastman Kodak Co.), 15 g PPO and 1.8 g dimethyl-POPOP (both, Packard Instrument Co.). Counting efficiency was 86 %. CsC1 in the gradient samples did not cause significant quenching.
Hydrolysis and analysis o[ polysaccharide Fractions comprising the polysaccharide peak of a given gradient were pooled, dialyzed against distilled water at 4 °, and lyophilized. The polysaccharide was tested for sensitivity to Qt-amylase (EC 3.2.1.1) (Worthington Biochemical Corp.) by incubation with the enzyme at 25 ° in 20 mM sodium phosphate (pH 6.9) corttaining 6 mM NaCI. The concentration of enzyme was 2/~g/ ml and that of polysaccharide, I.O mg/ml. The liberation of reducing groups was measured by reduction of 3,5-dinitrosalicylic acid. Hydrolysis for chromatographic analysis was carried out by dissolving the polysaccharide ill I M HCI to give 250/~g/ml and heating for I h at IOO°. The hydrolysate was dried over solid NaOH in a vacuum dessicator, twice washed with distilled water and redfied. Analysis for sugars was by methods modified from LEWIS AND SMITH11. Aqueous solutions of hydrolysate were applied to thin-layer cellulose chromatography plates (M2g-Polygram Cell 3oo, Brinkmann) and developed by two ascents with pyridineethyl acetate-acetic acid-water (36 : 36 : 7 : 21, by vol.) (Solvent system I.) Various sugars were chromatographed on the plates for comparison. Carbohydrate components were visualized by spraying with o.I M p-anisidine and phthalic acid in 95 % ethanol and heating. Radioactivity was located with a chromatogram radio-scanner (Model 6ooo-lo, Varian Aerograph). Radioactive material was recovered from nonsprayed spots by scraping the cellulose from the areas into distilled water. Glucose was determined with a glucose oxidase reagent (Glucostat Special, Worthington Biochemical Corp.).
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R. L. DOBSON, M. F. COOPER
Glucose was also enzymatically converted to gluconic acid using a method modified from BENTLEY AND NEUBERGER12. As a o.I % solution in IO mM sodium phosphate (pH 6.8), it was incubated at 22 ° with I/zg/ml glucose oxidase (EC 1.1.3.4) (Worthington Biochemical Corp.) and IOO/~g/ml catalase (EC 1.11.1.6) (Calbiochem) for 24 h. Products of this reaction were chromatographed on thinlayer silica gel plates (Polygram Sil N-HR, Brinkmarm). Two solvent systems were used, chloroform-methanol (60 : 40, by vol.) (Solvent system II) and butanol-acetic acid-water (5o : 25 : 25, by vol.) (Solvent system III). Radioactivity was located as before, and spots were visualized by spraying with 0. 5 % potassium permanganate in I ~][ NaOH. Comparison solutions were prepared with glucose (J. T. Baker Chemical Corp.) and gluconic acid (Sigma Chemical Co.).
RESULTS Fourteen mice were included in the feeding experiments, seven normally fed, one fasting, and six fasted-fed. The 48-h fast followed by refeeding was to ensure gluconeogenesis and stimulated glycogen synthesis at the time of tracer administration.
Glycogen labeling in normally/ed mice From E2-1iC]thymidine. Two normally fed animals were injected with E2-14C]thymidine and killed at 3 and 7 h. Gradient data from the 3-h mouse are presented in Fig. IA. The approx. 35/zg DNA present in the sample contains a total of 400 counts/min; the glycogen, amounting to 270/zg, contains a hardly detectable, estimated 4o counts/rain. Data from the 7-h animal were practically identical, except that the glycogen was not detectably radioactive. From [Me-14C]thymidine. One mouse received [Me-14Clthymidine and was killed at 2 h. The gradient pattern and DNA labeling were similar to those of the previous two animals. However, approx. IOO #g of glycogen in this case contained 500 counts/min, IO times greater than that in the glycogen of the [2-14C]thymidineinjected mouse of Fig. IA and more than was contained in the DNA. From E2-14C~thymine. Four animals were injected with E2-14C~thymine and killed at 3, 7 and 24 h. Gradient patterns were essentially the same as before, but DNA was not measurably radioactive and only the 3-h mouse showed detectable labeling of glycogen, estimated at 40 counts/rain as in the thymidine-injected mouse of Fig.iA. Thus, glycogen labeling was seen in normally fed animals only at the earlier times examined, 2 and 3 h after tracer injection. When x4C was in the 2-carbon position of the administered material, radioactivity appearing in polysaccharide was barely above the limit of detectability for the methods and sample-sizes used here. After thymidine with 14Cin the methyl group, however, it was an order of magnitude greater, and the amount of radioactivity in glycogen exceeded that in DNA.
Heavy labeling during active glucose and glycogen synthesis Of the six fasted-fed animals, three received radiothymidine and three radiothymine. They were killed between i and 6 h after injection. All six showed marked radioactivity of the polysaccharide. In Fig. IB are data from a 3-h E2-1aClthymidine-
Biochim. Biophys. Acta, 254 (1971) 393-4oi
397
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Fig. I. CsC1 d e n s i t y g r a d i e n t s of liver h o m o g e n a t e s f r o m mice in v a r i o u s n u t r i t i o n a l s t a t e s a t t h e t i m e t h e y were injected w i t h [l~C]thymidine or [14C]thymine. Main b a n d D N A IDNAm) a n d satellite (DNA~) d e t e r m i n e d b y A , 0 . m , p o l y s a c c h a r i d e (P) d e t e c t e d b y l i g h t - s c a t t e r i n g a t 32o n m a n d m e a s u r e d as h e x o s e b y d i p h e n y l a m i n e , a n d r a d i o a c t i v i t y as c o u n t s / r a i n are s h o w n p e r t o t a l u n d i l u t e d fraction. U l t r a c e n t r i f u g a t i o n a n d fraction a n a l y s e s were as described in MATERIALS AND ~[ETHODS. S e d i m e n t a t i o n is f r o m r i g h t to left. A. N o r m a l l y fed a n i m a l , 3 h a f t e r i n j e c t i o n w i t h [2-z*C]thymidine, ]3. F a s t e d - f e d a n i m a l , 3 h a f t e r b e i n g fed a n d injected w i t h [2-14CJthym i d i n e . C. F a s t e d - f e d a n i m a l , 4 h a f t e r b e i n g fed a n d injected w i t h [Me-Z4C]thymidine (note t h a t t h e r a d i o a c t i v i t y scale h a s b e e n c o m p r e s s e d io-fold). D. F a s t i n g a n i m a l , 2 h after injection w i t h [ 2 - " C ] t h y m i n e . E. F a s t e d - f e d a n i m a l , 2 h a f t e r b e i n g fed a n d injected w i t h [2-1*C]thymine. F. F a s t e d - f e d a n i m a l , 5 h a f t e r b e i n g fed a n d 4 h a f t e r i n j e c t i o n w i t h [2-1~C]thymine.
Biochim.Biophys.Acta,254
(1971) 3 9 3 - 4 o i
398
R. L, DOBSON, M. F. COOPER
injected mouse. The prominent radioactive peak in this gradient is glycogen, not DNA. The 560 pg present in the sample contains 20 times more radioactivity than is present in the 35 Pg DNA. While there is more polysaccharide than in Fig. IA, it represents glycogen that has been newly synthesized during the 3 h of refeeding, as liver glycogen stores are depleted by a 48-h fast (cf. Fig. ID). In fig. IC are the gradient data from a fasted-fed mouse injected, in this case, with EMe-14C]thymidine and killed 4 h later. While there is less glycogen than in Fig. IB, the labeling is an order of magnitude greater. The 325/~g polysaccharide in this gradient contains 200 times more radioactivity than does the 35 pg DNA. A similarly injected mouse, whose data are not shown, was killed after only I h. Its very much smaller amount of glycogen had the same specific activity as that in Fig. IC, suggesting that after receiving thymidine labeled in the methyl group, the fasted-fed mouse continues for some hours to incorporate 14C into glycogen in about the same proportion as during the first hour after injection. In Fig. ID are data from a fasting mouse killed 2 h after injection of [2-1ac]thymine, and in Fig. IE are data from a fasted-fed animal also killed 2 h after injection with the same material. No glycogen is detectable in the fasting mouse, and no radioactive labeling is seen associated with either the DNA or glycogen. The fastedfed animal, however, while showing no detectable radioactivity in DNA, shows a markedly radioactive glycogen peak. This represents polysaccharide synthesized during the 2 h of refeeding. The total radioactivity is almost identical to that in the similarly fasted-fed, but thymidine-injected, mouse of Fig. IB. An animal like that of Fig. IE, but sampled 6 h after injection, had a glycogen peak 4 times larger, indicative of the quantity of polysaccharide synthesized during the additional 4 h. The total radioactivity in the peak, however, was the same, suggesting that incorporation of 14C into glycogen from injected I2-14C]thymine is complete by 2 h. This contrasts with the more extended incorporation of radioactivity following injection of [Me-14Clthymidine noted above. Fig. I F shows data from a fasted-fed animal given food a full hour before injection with E2-14C]thymine, and killed 4 h after injection. Diphenylamine determinations of hexose are not available, but from the A3~0nravalues the amount of glycogen appears to be slightly more than that in Fig. IE. There is less labeling however, presumably because refeeding had produced a decrease in gluconeogenesis by the time of injection. A striking thing about this gradient is the position of glycogen well within the DNA region, emphasizing that it is not always separated from DNA even by ultracentrifugation in CsC1 gradients. Identification of radioactive glucose as the cause of glycogen labeling The polysaccharide isolated from CsC1 gradients responded to treatment with ¢¢-amylase by degradation as shown by a liberation of reducing groups. Chromatography of acid hydrolysates of the radioactive polysaccharide yielded a single spot with the staining properties of carbohydrate. It showed the specific color reaction of hexose 11. As seen in Fig. 2A, this spot had the same RF as glucose, and contained all the radioactivity. The eluate of the radioactive spot was positive for glucose when tested with the highly specific glucose oxidase reagent. A further stringent test of identity was applied. The eluted hydrolysate material of an unsprayed radioactive spot was reacted with glucose oxidase under conBiochim. Biophys. Acta, 254 (1971) 393-4Ol
399
RADIOACTIVE GLYCOGEN FROM INJECTED THYMIDINE A G
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Fig. 2. Thin-layer chromatogram and radio-scan tracings showing positions of stained sample spots and of radioactivity relative to origin (O) and solvent front (SF). Sample preparation and solvent systems were as described in MATERIALS AND METHODS. A. Glucose control (G) and hydrolysate of radioactive polysaccharide (H) on cellulose with Solvent system I, and radio-scan of H lane. B. Chromatogram on silica gel with Solvent system II of glucose control (G), control reaction mixture consisting of glucose with glucose oxidase and catalase (G + E), and the reaction mixture consisting of eluted hydrolysate spot from cellulose with glucose oxidase and catalase ( H + E ) , and radio-scan of H + E lane. C. Same as 13 except t h a t Solvent system III was used.
ditions that completely convert any glucose present TM, and rechromatographed. As seen in Fig. 2B and C, the product of the control glucose-enzyme reaction (G+E) remained at the origin with Solvent system II and showed a n R F of 0.2 5 with Solvent system III (as did gluconic acid when chromatographed with these systems). The product of the hydrolysate--enzyme reaction (H+E) behaved identically. The absence of any spot at the glucose position in both G + E and H + E lanes indicates that conversion of glucose was complete. All of the radioactivity behaved identically to glucose before reaction with glucose oxidase, and identically to the product, gluconic acid, afterwards, demonstrating that the 1'C was incorporated in glucose.
DISCUSSION
These experiments show that variable amounts of radioactivity can be incorporated into mouse liver glycogen after administration of radio-thymidine or thymine. This labeling can be vastly greater than that of DNA. Polysaccharide becomes especially radioactive when the tracer is injected at a time of active synthesis of both glucose and glycogen. The significance of this to studies involving DNA synthesis derives from the readiness with which polysaccharide can contaminate and be confused with nucleic acid 1-e. That confusion may occur even when isopycnic centrifugation methods are used for isolating DNA is illustrated by the overlap of DNA and glycogen shown in Fig. IF. Some investigators4 report banding positions similar to those in Fig. IA--E; Biochim. Biophys. Aaa, 254 (197 x) 393-4oi
400
R. L. DOBSON, M. F. COOPER
others s report glycogen to have precisely the same buoyant density as mouse DNA. The cause of variations in buoyant of density glycogen in CsC1 gradients is under study. How much error might radioactive glycogen introduce into estimates of DNA synthesis? Perspective on this question can be obtained from data in Fig. IC. The 320/~g of glycogen are seen to contain 9 ° ooo counts/rain, 28o counts/rain per #g. The 35/zg of DNA can be estimated to contain about 400 counts/rain, or I I counts/ rain per/zg. Now if, unbeknown to the investigator, this DNA were 35 % contaminated with glycogen (Note that M_~ARTINEZSEGOVIA e~ al. ~, using phenol extraction methods on mouse liver, reported persistent polysaccharide contamination of DNA in the ratio 0.54 : I.), then I #g of the DNA (containing I I counts/rain) would appear to contain 162 counts/rain. This would be a 14oo % error. Similarly, a io °/o contamination would produce an error of some 300 %, and a contamination as low as I o/ /o would lead to a 25 % erroi. If DNA is not significantly radioactive, as in the thymine-injected mice reported here (see Fig. IE), then any counts in glycogen mistakenly attributed to DNA could lead to even greater percentage errors. On the other hand, if similar considerations are based on data from a [2-14Clthymidine-injected mouse, as in Fig. IB, errors would be smaller b y a factor of IO than those based on Fig. IC. Thus a wide range of uncertainty m a y exist unless the investigator can be sure that this phenomenon plays no significant role in his experimental results. The liver, in regard to the effect under consideration, represents a case in which DNA synthesis is normally at a low level while glycogen metabolism is prominent. This would be expected to maximize the effect. Muscle might be somewhat similar, but the phenomenon should be less important in rapidly renewing cell systems. Studies on other tissues are in progress. That the mechanism of liver glycogen labeling is through incorporation of radioactive glucose, itself newly formed from thymine breakdown products, is apparent from the results reported here together with known features of thymidine catabolism 18, gluconeogenesis 14 and glycogen synthesis 15. The more intense and more sustained active labeling of glycogen after EMe-14Clthymidine than after E2-14C]thymidine is accounted for b y the fact that much of the a4C from the 2-carbon position is metabolized quickly to CO2 and can be lost, while that in the methyl group is metabolized to fl-aminoisobutyric acid and is more available for gluconeogenesis xe. Labeling of glycogen after EMe-14C!thymidine but not after E2-14C]thymidine has recently been reported in a protozoan 17. Our results accord with the findings of COUNTS AND FLAMM4 as regards magnitude of labeling following injection of I2-14C]thymine into normally fed animals and its decrease after the first few hours. However the m a x i m u m labeling they reported was Io-fold less than we observed in a normally fed mouse given EMe-14C]thymidine. It is likely that normal levels of gluconeogenesis and glycogen synthesis account for the relatively low-grade labeling seen in normally fed animals shortly after injection and that the decline after a few hours is due to mobilization of glucose from outer labeled tiers of glycogen molecules (c]. STETTEN AND STETTEN18).That such a decline was not observed in the fastedfed mice of these experiments is explained b y the radioactive glucose being incorporated into deeper portions of the newly synthesized glycogen molecules and therefore not so readily mobilized. Effects from natural diurnal variations in feeding and metabolic cycles are under investigation. Biochim. Biophys. Acta, 254 (1971) 393-4Ol
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The [Me-14C]thymidine-injected mouse of Fig. IC incorporated 3 % of the administered radioactivity into liver glycogen. The [2-14C]thymidine-injected animal of Fig. I B incorporated 0. 3 %. The latter figure is about the same as that which can be derived from the data of SCHNEIDER AND GRECO 19 for total incorporation observed in various fractions of liver particulate preparations, including lipid, protein, DNA, RNA, and acid-soluble, after injection of EMe-3H]thymidine and [2-14C~thymidine. The fact that the mouse of Fig. IC incorporated IO times more than this into liver glycogen alone illustrates how greatly the labeling of non-DNA components~ glucose and glycogen in particular, can depend upon metabolic states at the time of radiothymidine administration.
ACKNOWLEDGMENT
The authors wish to thank Dr. Frederick T. Hatch and Joseph A. N[azrimas for helpful suggestions during the course of these experiments and for review of the manuscript. This work was performed under the auspices of the U.S. Atomic Energy Commission. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Atomic Energy Commission or of the University of California to the exclusion of others that may be suitable. REFERENCES I O. T. AVERY, C. M. MACLEOD AND I~. McCARTY, J . Exp. Med., 79 (1944) 137. 2 J. MARMUR, J. Mol. Biol., 3 (1961) 2o8. 3 Z. M. ~ARTINEZ SEGOVIA, F. SOKOL, I. L. CvRAVESAND W. W. ACKERMANN, Bioehim. Biophys. Acta, 95 (1965) 329. 4 W. :B. COUNTS AND W. G. FLAMM, Biochim. Biophys. Acta, 114 CI966) 628. 5 L. PIK6, A. TYLER AND J. VINOGRAD, Biol. Bull., 132 (1967) 68. 6 D. G. PRITCHARD, R. ~¢~. HALPERN AND R. A. SMITH, Biochim. Biophys. Acta, 338 (1971) 127. 7 L. PIK6, Devel. Biol., 21 (197o) 257. 8 R. L. DOBSON AND M. F. COOPER, m a n u s c r i p t in preparation. 9 F. DEVIK AND K. HALVORSEN, Nature, 197 (1963) 148. i o L. O. CHANG AND W. B. LOONEY, Cancer Res., 25 (1965) 1817. I I B. A. L E w i s AND F. SMITH in E. STAHL, Thin-Layer Chromatography, Springer-Verlag, N e w York, 1969, p. 8o 7. 12 R. BENTLEY AND A. NEUBERGER, Biochem. J., 45 (1949) 584 . 13 J" E. CLEAVER, Thymidine Metabolism and Cell Kinetics, N o r t h Holland, A m s t e r d a m , 1967, P.54 14 H. A. LARDY, The Harvey Lectures, Series 6o, Academic Press, New York, 1966, p. 261. 15 H. Cy. HERS AND H. DEWULF, Syrup. on Control o[ Glycogen Metabolism, Proc. 4th Meeting Fed. Eur. Biochem. Socs., Oslo, I967, Universitetsforlaget, Oslo, 1968, p. 65. 16 K. FINK, R. E. CLINE, R. B. HENDERSON AND R. •. FINK, J. Biol. Chem., 22i (1965) 425 . 17 P. A. LANZETTA AND J. BERECH, JR., Fed. Proc., 3° (1971) 1124. 18 M. R. STETTEN AND D. STETTEN, JR., J. Biol. Chem., 213 (1955) 723 • 19 ~V. C. SCHNEIDER AND A. E. GRECO, Bioehim. Biophys. Acta, 228 (1971) 61o.
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