Nucleic Acid Metabolism in Regenerating Rat Liver VI. Soluble Enzymes Which Convert Thymidine to Thymidine Phosphates and DNA*T

Home , Nucleic acid metabolism

Nucleic Acid Metabolism in Regenerating Rat Liver VI. Soluble Enzymes Which Convert Thymidine to Thymidine Phosphates and DNA*t

F J. BOLLUM~ AND VAN" R. POTTER

(McArdle Memorial Laboratory, University of Wisconsin, Madison 6, Wis.)

The presence of a multienzyme system which The time of sacrifice was recorded and referred to as the incorporates thymidine into DNA in the soluble time at which enzyme activity was sampled. Orotic acid incorporations in trivo were recorded for time points corre- fraction of regenerating rat liver homogenates sponding to 1 hour before death, i.e., the mid-point of the (5, 6) raises the question of the biological sig- 9,-hour period of incorporation. When in vivo incorporation nificance of this enzyme system. The rate of DNA was not studied, the supernatant fraction (no radioactive synthesis in vivo as a function of time after partial uridine nucleotides present) could be used for either kinase or polymerase assay. hepateetomy has been studied in several labora- Assay for kinase.--To assay the supernatant fraction for tories, with a variety of isotopic substrates (1, capacity to phosphorylate thymidine, chromatographic pro- 11-13, and references cited). To examine the re- cedures were employed to separate the products for analysis. lation between DNA synthesis in vivo and activity Although Dowex-1 columns were useful for such assays, it of the soluble enzyme system, a technic for study- was usually more convenient to use paper chromatography. Two solvent systems were employed: (a) the isobutyric: ing these phenomena simultaneously in the same NHa:EDTA 1 solvent of Krebs and Hems (15) and (b) iso- animal was used, following the plan previously propanol: acetic acid: H20 (170:10: 70). In both solvents lg~ used to correlate DNA synthesis in vivo and in is inversely proportional to level of phosphorylation, but tissue slices (13). The results have shown that adenine nucleotides move faster than thymidine nucleotides in solvent (a) and slower in solvent (b). For routine incubations the appearance of the enzyme system is correlated 50/A. of the supernatant fraction, ~5 ~1. of a substrate mixture, with the appearance of DNA synthesis in vivo, and 50 #1. 0.~ M Tris, pH 8.0, were incubated at 88~ C. The but that enzymatic activity continues to increase substrate mixture was made up to provide the following sub- over the period 24-48 hours after hepatectomy, stances and final concentrations: ATP, 5 raM; H3-thymidine whereas DNA synthesis in vivo decreases. (39 ~c/~mole), .06 raM; 8-phosphoglycerate, 6 m~; and Mg ++, 5 raM. Reactions were stopped by cooling and addition of 10 ~1. of 100 per cent (w/v) TCA. The protein precipitate MATERIALS AND METHODS was centrifuged, and an aliquot (10-20 ~1.) of the clear super- Male albino rats weighing 140-180 gm. were partially natant fluid was applied to Sleicher and Schuell No. 589 hepatectomized and fed ad libitum. At the desired time after blue ribbon filter paper for chromatography in one of the sol- hepatectomy each animal received 1 ~mole of orotic acid-6-C 14, vents above. It was desirable to add markers of TMP and specific activity 1 ~e/~mole. Exactly ~ hours later the animal thymidine to the reaction mixture before deproteinization was sacrificed by decapitation and the liver removed and in order to aid in cutting the strip prior to elution and counting. placed in ice-cold saline; ~0 per cent homogenates were pre- TTP and TDP were found by their positions relative to pared with the use of the buffered sucrose homogenization ATP and were tentatively identified by Rr (17) and by acid medium described previously (6). Nuclei were removed by hydrolysis to a compound traveling with authentic TMP centrifuging homogenates for 10 minutes at 600 X g, and the on paper and column chromatograms. Compounds were eluted specific activity of the DNA isolated from them was determined from paper with water, and aliquots were plated on A1 planch- by the usual procedure (12). The nuclear supernatant fraction ets. Because of the .qmall amount of material on the planchet, was then centrifuged for 60 rain. at 44),000 r.p.m. (-- 105,000 )< no corrections could be made for self-absorption, and therefore g) in the Spinco preparative centrifuge. Although the clear this technic is rather crude when tritiated substrates are supernatant fraction contained radioactive uridine nucleotides, used. The differences observed were of a magnitude which these did not interfere with the polymerase assays (see below). could be interpreted in a semiquantitative manner. The most * This work was supported in part by a grant (No. C-646) useful value obtained from the chromatograms was per cent from the United States Public Health Service. 1 Abbreviations as follows: EDTA, ethylene diamine tetra A preliminary account of this work has been published (5). acetate; ATP, adenosine triphosphate; TCA, trichloroacetic :~ Postdoctoral Fellow of the National Cancer Institute, acid; DNA, deoxyribonucleic acid; TMP, 5'-thymidylic U.S.P.H.S., 1956-1958. Present address: Biology Division, acid; TDP, 5'-thymidlne diphosphate; TTP, 5'-thymidine tri- Oak Ridge National Laboratory, Oak Ridge, Tennessee. phosphate; dAMP, deoxyadenylic acid; dCMP, deoxycytidylic acid; dGMP, deoxyguanylic acid; H3-TMP, tritium-labeled Received for publication February s 1959. thymidylic acid; Tdr, thymidine; DNAse, deoxyribonuclease. 561

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1959 American Association for Cancer Research. 56~ Cancer Research Vol. 19, June, 1959 of total radioactivity present as phosphorylated compounds, RESULTS and this was used as a measure of thymidine kinase. Assay for polymerase.--Routine incubations contained 0.1 The capacity of regenerating liver supernatant ml. DNA (500 #g.), 0.2 ml. supernatant fraction, and 0.2 fraction to form thymidine nucleotides is shown ml. of a substrata mixture. The substrate mixture was prepared in Chart 1. These data were obtained from a to contain in the final incubation mixture: ATP, 5 mM; Mg ++, series of paper chromatogrtims such as the repre- 5 raM; 3-phosphoglycerate, 6 raM; Tris: HCI, pH 8.0, 40 mM; dAMP, dGMP, dCMP, 0.1 mM; Ha-thymidine (39 ttc/gmole), sentative one depicted in Chart 2. A similar study 0.06 raM. DNA was isolated as described previously (12). with supernatant fraction from normal liver indi- An aliquot was plated, and an aliquot was taken for calori- cated insignificant formation of phosphorylated metric analysis (7). The specific radioactivity of DNA after 60 minutes of incubation is proportional to enzyme activity (6) in this assay. Although protein determinations were per- 4OOO 24hr. regen., 60' formed (9) on the supernatant fraction from each animal, TP the agreement between samples was within the limit of the precision of the method, and therefore specific activities are J~ TMP Tdr not corrected for small variations in protein concentration. Protein concentration of the supernatant fraction as prepared w 20o0 i i I I ! II o H3-Tdr-.-~ H3-TMP.-~ H3-TTP I 24 hr. regenerating rot Liver ($3)~ I000

._ I"I ~40 I 0 5 I0 15 20 25 30 Q: Centimeters CHART 2.--Phosphorylation of thymidine in presence of soluble enzyme from regenerating rat liver. Paper chromato- g ram of 60-minute incubation; isobutyric: NH3: ethylenedia- 2O minetetracetic acid solvent. Abbreviations as in Chart 1. o 3 weeks regen. IOOC ! I "-"I I0 20 30 24hr. regen. -L Minutes Incubation "~ !006 38 ~ J L, Cm~RT 1.--The formation of thymidine nucleotides by Q Normal 24-hour regenerating rat liver supernatant fraction. Abbrevia- ! tions: H3-Tdr, tritium-labeled thymidine; TMP, TDP, and I000 TTP, thymidine mona-, di-, and triphosphate; $8, soluble supernatant fraction of liver cytoplasm ic fraction. r V ""-1 ,, 0 5 !0 15 20 25 30 35 40 here averaged 19 mg/ml. When supernatant fractions contain- Oentimeters ing radioactive pyrimidine nucleotides (from the arctic acid injection) were used, suitable controls were run. Such controls CHART 3.--Phosphorylation of thymidine in presence of contained all the components listed for the enzyme assay soluble enzyme from regenerating rat liver. Representative except H3-thymidine. In aU cases the radioactivity incorpo- chromatograms of 10-minute reaction mixtures from super- rated into DNA was negligible. This was not unexpected, natant fraction at several stages of regeneration; isobutyric since even if the conversion of ribotide to deoxyribotide acid: NH3: ethytenediaminetetracetic acid solvent. The major demonstrated by Grossman (10) were operating in such super- peaks are TTP + TDP, TMP, and unchanged Tdr, respective- natants, the folic-dependent methylation of deoxyuridine (2, ly, from left to right. 8, 16) would probably be ineffective in the reaction system employed. compounds from thymidine of the specific activity All radioactivity determinations were performed in window- employed in these experiments. The appearance less flow counters (6). Since DNA plates from enzyme incuba- and disappearance of the thymidine kinase are tions all contained essentially the same amount of DNA, further demonstrated in Chart 3, which shows rep- no correction for self-absorption has been made. C 14 deter- resentative chromatograms on supernatant frac- minations on DNA labeled in viva after injection of labeled arctic acid were corrected for self-absorption in the usual tion assays for normal, ~4-hour regenerating, and way (12). completely regenerated (3-week) rat livers. The

chromatograms of Chart 3 are part of a larger in the period of ~4-30 hours, when DNA synthesis study designed to find out whether a sequential measured by orotic acid incorporation in vivo induction of enzymes was occurring, i.e., could is decreasing and enzyme activity for thymidine thymidine kinase be shown to increase significantly in vitro is increasing. Aside from the period of before the polymerizing enzyme appeared? The ~4-30 hours, there is a remarkable parallelism complete study is presented in Table 1, which between the two curves, including the period of contains results of kinase and polymerase assay initial rise (18-~4 hours), the period of secondary on the same supernatant fraction. These results fluctuations (30-50 hours, cf. 1~), and the final indicate the simultaneous increase of two of the period of decline to resting values. enzymes, thymidine kinase and polymerase, re- That the polymerizing enzyme is actually in- quired for the incorporation of thymidine into creasing is shown in Table ~, in which the time DNA. Although finer discrimination in methodolo- points were obtained by the method described gy and timing might detect some difference, this above, which starts with thymidine, and by an conclusion is valid within the limits of the present assay (Method III, [6]) which utilizes preformed study. Studies with H a-TMP will be helpful in assaying the activity of enzymes that phosphory- TABLE 1 late thymidylic acid. The earlier study of Hecht, THE APPEARANCE OF THYMIDINE rHOS- Potter, and Herbert (14) indicated that TMP PHORYLATING ENZYMES AND POLYMERIZING is phosphorylated by cytoplasmic fraction from ENZYME IN EARLY REGENERATION regenerating liver. Per cent of Time after partial thymidine 9 . :5 Polymerase t I hepatectomy phos- =O I phorylated* Unoperated 7.6 40 ,s 9 2 ;2 Unoperated 7.3 460 =o. Unoperated 11.0 950

12 hours 9.1 780 12 " 9.3 200 17 " 15.0 1,660 17 " 5.7 440 23 " 70 11,000 23 " 56 10,600 27 " 51 12,500 i m 3 weeks 10 35 0 20 40 60 80 iO0 120 8 . 8.6 70 HOURS AFTER PARTIAL HEPATEGTOMY *Ten-minute incubation, total of TMP, CHANT 4.--Correlation curve: orotic acid incorporation TDP, and TTP. in vivo and thymidine incorporation in vitro. Data from three t Specific activity of DNA after 60-minute separate experimenls with a total of 44 rats are plotted. In incubation. all cases reported, both assays were carried out for each animal. Each point is the average of two to four rats, with deoxynucleoside triphosphates and therefore meas- the larger number at the steepest parts of the curves. ures polymerase independently of kinases. The increase in polymerase activity is observed by It is of some interest at this point to mention both methods. It is noteworthy that the actual that the enzymes which phosphorylate dAMP, level of polymerase in normal liver or early re- dGMP, and dCMP are readily detectable in super- generation is appreciably higher than is indicated natant fractions from normal as well as regenerat- by the assay which utilizes thymidine. Since this ing rat livers. It is also noteworthy that the specific finding concerns the early phase of regeneration, activities (#moles product/hour/rag protein) of it does not affect conclusions which concern the these enzymes are much greater than the cor- simultaneous increase of enzyme activity and DNA responding thymidine-converting enzymes (4). synthesis, and this correlation at later times of The complete curve demonstrating the correla- regeneration. tion between enzymatic incorporation of thymidine and orotic acid incorporation in vivo is illus- DISCUSSION trated in Chart 4. It is apparent that the forms The major objective of this investigation was of these two curves are remarkably different in to observe the activity of the soluble polymerizing certain respects. The most striking difference is enzyme during a period when the rate of DNA

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1959 American Association for Cancer Research. 564 Cancer Research Vol. 19, June, 1959 synthesis is undergoing change. The result shows not at this time be considered as establishing that the enzyme (system) appears in the super- the site of DNA synthesis as it occurs in the celt. natant fraction at the time DNA synthesis begins In particular, the tow level of thymidine kinase and constitutes reasonable evidence that ~the two in soluble fraction from normal animals is a subject phenomena are physiologically related. The rela- for further research, since a rather large percentage tion between enzyme activity and rate of DNA of normal whole homogenate incubations has re- synthesis beyond the time of simultaneous ap- sulted in the appearance of thymidine nucleotides. ~2 pearance remains unclear. The close correlation Our earlier speculation (3) that enzyme activity between slices and in vivo incorporation reported may reflect the total capacity for DNA synthesis previously (13) is not 'incompatible with the lack (cells synthesizing DNA and cells having syn- of detailed correlation observed in this study. thesized DNA and still retaining this enzymatic The previous correlation was found when both capacity), whereas in vivo incorporation measures parameters were determined at a cellular level, only the activity of cells actually synthesizing and the present study compares cellular (con- DNA during the interval of exposure to isotope, remains as a useful working hypothesis. Although TABLE the sudden increase in activity of two enzymes DEMONSTRATION OF POLYMERASE of DNA synthesis cannot immediately be inter- APPEAR&NCE BY USE OF ~INASE- preted as enzyme induction, it is tempting to INDEPENDENT ASSAY* speculate that such might be the case. Further studies, including demonstration of new protein

I:[OLlr s Kinase- Kinase- formation, will be required in making this decision. post- dependent independent Procedures for obtaining purified enzyme (4) will operative assay assay be necessary for such studies. 0 370 2,340 The crude supernatant fraction of 48-hour re- 0 24O 2,720 generating liver has a specific activity of approxi- t0 220 1,670 10 100 1,960 mately 0.2 mgmoles HS-thymidine incorporated/ 18 830 2,920 mg protein/hour. By the assumption that equi- 18 1,500 $ ,720 molar proportions of each nucleotide are incor- 24 8,650 9,030 ?.4 7,500 9,700 porated into DNA, the total supernatant fraction 48 6,950 8,450 has a maximum capacity to synthesize about 48 5,240 6,800 72 7,850 12,600 30 gg. of DNA per hour. In the intact rat, liver regeneration results in synthesis of approximately 10 rag. of DNA in 5 days (Chart 7, Paper I All data are specific activity of DNA after 60 minutes' incubation at 38 ~ C. [IS]). It is suggested that the supernatant enzyme * In the kinase-dependent assay thy- has a capa~ty approximating DNA synthesis in midine is utilized as substrate. In the vivo sufficient closely to be the enzyme responsible kinase-independent assay TTP is utilized as substrate and is not limited by the rate for DNA synthesis. of conversion of thymidine to TTP. SUMMARY trolled) DNA synthesis with enzymatic (uncon- 1. A technic for comparing DNA synthesis in trolled) activity. Perhaps whole homogenate and regenerating rat liver in vivo and in cell-free super- ceil fractionation studies wilt be helpful in further natant fractions from the same animal in vitro resolution of this issue. The conclusions to be has been devised. drawn from the present investigations are some- 2. Methods for evaluating the activity of en- what limited by the fact that only one celt fraction zymes phosphorylating thymidine are presented. was examined in detail. The methods developed 8. Enzymatic incorporation of thymidine into and the activity levels observed in the supernatant DNA was found to appear at the same time traction do, however, provide a base-line for fur- (18 hours after operation) as DNA synthesis in ther detailed studies on other celt fractions. Cell vivo but continued to increase over the period fractions which contain appreciable phosphatase, of 18-30 hours while DNA synthesis/n v~vo was pyrophosphatase, and DNAse activity will require decreasing (s hours). more elaborate methodology. Some of the details 4. The enzymes which convert thymidine to presented in this communication are concerned thymidine nucleotides appear at about the same primarily with the demonstration of polymerizing time as the potymerizing enzymes. activity in the supernatant fraction and should 2 Unpublished experiments, Miss Anne Brumm.

ACKNOWLEDGMENTS McELRoY and B. GLASS (eds.), The Chemical Basis of We wish to thank Miss Ellen Herman and Miss Katherine Heredity, pp. 609-14. Baltimore: Johns Hopkins Univ. Roberts for technical assistance. Radiochemical analyses were Press, 1957. performed by Mrs. Lynne Gilboe and Mrs. Dorothy McManus, 9. GORNALL, A. G.; BARDAWlLL, C. J.; and DAVID, M. under the general supervision of Dr. Charles Heidelberger. M. Determination of Serum Proteins by Means of the Biuret Reaction. J. Biol. Chem., 177:751-66, 1949. REFERENCES 10. GROSSMAN, L. Conversion of Purine and Pyrimidine Ribo- 1. ANDERSON, E. P., and AQUIST, S. Further Studies on nucleotides and Ribonucleosides to Deoxyribonucleotides the Turnover of Polynucleotides in Regenerating Rat and Nucleosides. Fed. Proc., 17:235, 1958. Liver. Acta Chem. Scandinav., 10:1576-82, 1956. 11. HAMMARSTEN,E.; AQUIST, S. ; ANDERSON,E. P.; ELIxssoN, 2. BLAKELEY, R. L. Methylation of Uracil Deoxyriboside N. A.; and THORELL, B. The Turnover of Polynucleotides by Soluble Enzymes of Thymus. Biochim. et Biophys. and Proteins in Regenerating Rat Liver, Studied with acta, 24:224, 1957. Glycine-N15. Acta Chem. Scandinav., 10:1568-75, 1956. 3. BOLLUM, F. J. DNA Synthesis by Enzymes from Mam- 12. HECHT, L. I., and POTTER, V. R. Nucleic Acid Metabolism malian Tissues. Fed. Proc., 17:193, 1958. in Regenerating Rat Liver. I. The Rate of Deoxyribo- 4. ~. Incorporation of Thymidine Triphosphate into nucleic Acid Synthesis in Vivo, Cancer Research, 16: Deoxyribonucleic Acid by a Purified Mammalian Enzyme. 988-93, 1956. J. Am. Chem. Soc., 80:1766, 1958. 13. ~. Nucleic Acid Metabolism in Regenerating Rat 5. BOLL~rM, F. J., and POTTER, V. R. Incorporation of Thy- Liver. V. Comparison of Results in Vivo and in Tissue midine into DNA in a Supernatant Fraction from Rat Slices. Ibid., 18:186-92, 1958. Liver Homogenates. Abstr., Am. Chem. Soc., 132d Meet- 14. HECHT, L. I.; POTTER, V. R. ; and HERBERT, E. In Vitro ing, New York, 19-C, 1957. Phosphorylation of Pyrimidine Deoxyribosemononucleo- 6. ~. Incorporation of Thymidine into Deoxyribonucleic tides. Biochim. et Biophys. acta, 15:134-35, 1954. Acid by Enzymes from Rat Tissues. J. Biol. Chem., 15. KaEBS, H. A., and HEMS, R. Some Reactions of Adenosine 233: 478--82, 1958. and Inosine Phosphates in Animal Tissues. Biochim. et 7. DISCHE, Z. l~ber einige neue charakteristische Farben- Biophys. Acta, 19.: 172-80, 1953. reaktionen der Thymonukleins~ure und eine Mikrometho- 16. PHEAR, E., and GREENBERO, D. M. The MethyIation de zur Bestimmung derselben in tierischen Organen mit of Deoxyuridine. J. Am. Chem. Soc., 79:3787--41, 1957. Hilfe dieser Reaktionen. Mikrochimie, 8:4-$2, 1930. 17. POTTER, R. L.; SCHLESINGER, S.; BUETTNER-JANUSCH, 8. ~qIEDKIN~ M., and KORNBERO, A. The Enzymatic Con- V.; and THOMPSON, L. The Isolation of Pyrimidine Deoxy- version of Deoxyuridylic Acid to Thymidylic Acid and nucleotides from the Acid-Soluble Extract of Thymus. the Participation of Tetrahydrofolic Acid. In: W. D. J. Biol. Chem., 226:381-94, 1957.

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1959 American Association for Cancer Research. Nucleic Acid Metabolism in Regenerating Rat Liver VI. Soluble Enzymes Which Convert Thymidine to Thymidine Phosphates and DNA

F J. Bollum and Van R. Potter

Cancer Res 1959;19:561-565.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/19/5/561.citation

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/19/5/561.citation. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.