Showdomycin, a New Nucleoside Antibiotic'

[CANCER RESEARCH 28, 1106-1610,Aug@at1968] Showdomycin, A New Nucleoside Antibiotic'

S. Roy-Burman,P.Roy-Burman,and D. W. Visser Department of Biochemistry, University of Southern California, School of Medii*ie, Los Angeles, California 9008$

SUMMARY Streptococcus pyogenes (14). Showdomycin has antitumor activity against Ehrlich ascites cells both in vitro and in vivo The metabolism and inhibitory effects of the carbon-linked and is active against cultured HeLa cells ( 11, 14, 15) . Its nucleoside antibiotic showdomycin were investigated in cell structure has been established as 3-$-o-ribofuranosylmaleimide free preparatiozis of Ehrlich ascites cells. The antibiotic is not (5), a carbon-linked nucleoside antibiotic. a substrate for nucleoside kinase or nucleoside phosphorylase. The structural similarity of showdomycin, uridine, and Showdomycin shows inhibitory effects on certain enzymes in an pseudouridine are apparent from the formulae (Chart 1). Ehrlich ascites cell preparation, which are involved in uridine Darnall et at. (5) have pointed out that showdomycin may be and orotic acid metabolism. It inhibits Ã¼ridine-5'-monophos viewed as pseudouridine which has lost an -NH group in the phokinase, uridine phosphorylase, and possibly orotidylic acid contraction to a five-membered ring. pyrophosphorylase reactions, but has no effect on the activity The unique structure of showdomycin, its structural simi of uridine kinase or adenosine phosphorylase. Showdomycin larity to uridine, and its biologic properties prompted this in strongly inhibits bovine liver uridine-5'[email protected] vestigation of its metabolism and its effect on various enzymes, dehydrogenase but has no effect on rabbit muscle lactic acid particularly those involved in uridine metabolism. dehydrogenase. The selective inhibition of certain enzymes by showdomycin may be related to the alkylating property of its maleimide structure, which is known to specifically react with MATERIAL AND METHODS sulfhydryl groups. Showdomycin was obtained as a gift from Dr. Ronald K. Robins of the University of Utah, Salt Lake City, Utah, and INTRODUCflON Dr. Haruo Nishimura of Shionogi Research Laboratory, The antibiotic, showdomycin, first isolated by Nishimura Fukushima-Ku, Osaka, Japan. The purity of the compound et at. (15) from Streptomyces showdoensis, inhibits growth of was determined in two descending paper chromatographic several Gram-positive and Gram-negative bacteria and is par systems. The compound migrated as a single ultraviolet ab ticularly active against Streptococcus haemotyticus (15) and sorbing area in ethanol 0.5 M ammonium acetate, pH 6.5 (5:2 v/v) and isopropanol: concentrated HC1: 1120 (65:16.7:18.3 v/v/v) solvent systems, the respective RF values being 0.64 1 This investigation was supported by research grants from and 0.68. Uridine-2-14C and UMP-2-'4C2 were purchased from USPHS (CA 02373-12), the American Cancer Society (E-412) Schwarz BioResearch, Inc. Orotic acid-6-14C was obtained and (in part) by a Dernham Senior Fellowship of the American Cancer Society, California Division (No. D120). from New England Nuclear Corporation. Nucleoside, nucleo Received January 26, 1968; accepted April 21, 1968. tides, ribose-5'-phosphate, and 3-phospho-D-glyceric acid (sodium salt) were obtained from California Corporation for Biochemical Research. A supernatant fraction (100,000 x g) from a hyperdiploid HN strain of Ehrlich ascites tumor cells was prepared as described previously (18, 21). A crude preparation of uridine phosphory 0 _.-@ lase was obtained from Ehrlich ascites cells in the following manner. Ascites cells were washed as described previously (21) and disrupted using a French press at 1800 lb/sq inch at HOC@ HOCH2 0 0-5Â°C in 2 volumes of buffer containing 0.05 M potassium phosphate, pH 7.4, and 0.13 M potassium chloride. An addi tional amount of buffer was added to the disrupted cells to make OH OH OH OH

2 Abbreviations used are : UMP, uridine-5'-monophosphate; UDP, uridine-5'-diphosphate ; UDP-glucose, uridlne-5'-diphoe SHOWDOMYCIN URIDINE PSEUDOURIDINE phate-a-n-glucose ; UTP, uridine-S'-triphosphate ; Tris, tria(hy droxymethyl)aminomethane ; NAD+, nicotinamide adenine di Chart 1. Graphic formulas. nucleotide.

AUGUST 1968 1605

a final concentration of 3 ml of buffer per gm of cells. The omitted from the incubation mixture. The detection limit of suspension was centrifuged for 60 min at 85,000 x g. The the photometric assay procedure is about 10 optical density supernatant enzyme preparation contained 12-14 mg protein/ units which represents about 1% of the added showdomycin. ml and had a specific activity of 110-160 (17). This prepara The fractions were also tested with Elson-Morgan's color re tion was used for assay of uridine phosphorylase and adenosine agent, which gives a red color with showdomycin (15). The phosphorylase. results, with and without showdornycin, were identical based Preparation of acid-soluble fractions from the various incu on these assay procedures, showing that showdomycin is not. bation mixtures and separation of phosphorylated derivatives converted to phosphorylated derivatives by enzyme prepara by chromatography on Dowex 1 (formate) were performed tions which converted 50-70% of uridine-'4C to UTP. as described previously (18). Effect of Showdomycin on Uridine Metabolism. The results Uridine diphosphate glucose dehydrogenase was purchased of experiments designed to determine the effect of showdomy from Sigma Chemical Company and was assayed (19) by cin on the conversion of uridine-'4C to phosphorylated deriva following NAD+ reduction. Rabbit muscle lactic dehydrogenase tives are shown in Table 1. The data show that showdomycin was obtained from California Corporation for Biochemical did not inhibit uridine kinase but strongly inhibited the con Research and was assayed by determining the rate of oxidation version of UMP to UDP. The latter effect increased with in of reduced NM) with pyruvate as substrate. creasing concentrations of showdomycin. In the presence of 2.5 @molesshowdomycin per ml reaction mixture, UMP-14C RESULTS represented more than 70% of the radioactive nucleotides, whereas in the absence of showdornycin, about 90% of the Effect of Kinases on Showdomycin. Since inhibitory effects acid-soluble nucleotides was recovered as UTP. Uridine kinase of most nucleoside derivatives are dependent upon a prior activity was not inhibited by showdomycin under the same conversion to phosphorylated derivatives, it was of interest to conditions. determine the susceptibility of showdomycin to kinase activi Since the previous experiments indicated that showdornycin ties. Showdomycin was incubated at pH 7.8, 7.0, and 6.2 with specifically inhibited UMP kinase, the effect of the analog on a supernatant fraction prepared from Ehrlich ascites cells the phosphorylation of UMP-14C was investigated. In the having high levels of uridine, UMP, and UDP kinase activity presence of 1.6 @molesshowdomycin per ml reaction mixture, (18, 21) . Incubation mixtures were treated as described pre UTP formation was inhibited about 50% (Table 2). When viously (18) except that the separation of acid-soluble corn the enzyme mixture was preincubated with the same concen ponents was carried out on a Dowex 1 (chloride) column tration of showdomycin prior to the addition of UMP-14C, using a gradient concentration of hydrochloric acid and sodium UMP kinase activity was completely inhibited, whereas 94% chloride. Initially the mixer contained 1 liter of water and the of UMP was converted to the triphosphate in the absence of reservoir contained 0.01 N hydrochloric acid. After collection showdomycin. Preincubation of showdomycin with the reaction of about 1 liter of eluent, the solution in the mixer was changed mixture prior to the addition of both UMP-'4C and enzyme did to 1 liter of 0.01 N hydrochloric acid and that in the reservoir not increase the inhibitory effect. to 0.2 M NaC1. After collection of about 2 additional liters of Effect of Showdomycin on the Conversion of Orotic Acid eluent, the column was eluted directly with 0.5 M NaCl in 0.01 to Uridine Nucleotides. Showdomycin inhibited the conversion N hydrochloric acid. Since showdomycin in acidic solution has of orotic acid-'4C to uridine nucleotides (Table 3) . At a con @ a characteristically high absorbance at 220 and a very low centration of 0.56 @&moleshowdomycin per ml reaction mixture, absorbance at 260 m@ ( 15) , the absorbance of the effluent was about 50% of the total radioactivity was recovered as erotic measured at 220 m@ and compared with a control which was acid, whereas in the absence of showdomycin, less than 15% handled in an identical manner, except that showdomycin was of the radioactivity remained as orotic acid, the remainder

Table 1

%UMPUDP recovered as Showdoinycin phos added phorylated (@unoles)Radioactivityderivatives00.5088.670.180.05.029.305.050.084.310.063.302.720.686.6sugarUDPUTPTotal

Effect of showdomycin on uridine-14C phosphorylation. The reaction mixtures (4 ml) con tamed : 20 @molesATP ; 10 @@molesphosphoglycericacid ; 50 @@molesMgCl2; 250 @&moles tris(hydroxymethyl)aminomethane-HC1, pH 7.5; 1 @moleuridine-14C (7.6 x 105 cpm) ; 5 or 10 @imolesshowdomycin ; and 22 ml (29 mg) 100,000 x g supernatant fraction. Incubations were carried out for 10 mm at 37Â°Cand terminated with 1.0 ml 4.0 M perchloric acid. The radioactive nucleotides in the acid-soluble fractions were separated as described in the text. Radioactivity of total acid-soluble components prior to chromatography was taken as 100% in each experiment. UMP, uridine-5'-monophoaphate; UDP, uridine-5'-diphosphate; UTP, uridine-5'-triphosphate.

1606 CANCER RESEARCH VOL.28

Table 2 Table 4

Showdomycin%UMPTJDPUTP0UMP-'@C0394.6593.905aNoneafter recoveredas (manoles)Without added (i@moIes)AdditionpreincubationRadiosctivity Addition (jimoles)Ribose-1-phosphateformed preincubationPreincubstionUridineShowdomycin1.0031.02012202.71.00.6192.05921.012107.90.91.01.982.00.91.02271.01.05.346.01.010.053.0

UMP-'@C,[email protected]@14C98.7000

Effect of showdomycin on UMP@14C phosphorylation. The re action mixture (3 ml) contained : 7 @molesATP ; 20 jimoles phos phoglyceric acid ; 33 @molesMgCl2; 250 @molestris(hydroxy methyl)aminomethane â€¢HC1,pH 8.0; 1 @moleUMP_14C (1 x 10@ Effect of showdomycin on uridine phosphorylase activity. The cpm) ; 5 or 10 @molesshowdomycin; and 1 ml (13 mg) 100,000 x reaction mixtures (0.15 ml) contained : 15 @@molespotassiumphos g supernatant fraction. The reaction mixtures were incubated for phate buffer, pH 7.4; 0.1 ml (1.4 mg) enzyme preparation; and 10 mm at 37Â°Cin the absence of UMP-'@C or both UMP@14Cand the indicated amounts of showdomycin. After preincubation of enzyme as indicated and after their addition incubation was con the reaction mixtures for 10 min at 37Â°C,1 @&moleuridine was tinued for an additional 10 min. In experiments where preincuba added and the incubation was continued for 20 mm. In experi tion was not carried out, UMP@14C and the enzyme were added ments where preincubation was not carried out, uridine was added to the reaction mixture at zero time and incubations were carried to the reaction mixture at zero time and incubations were carried out for 10 mm. Other conditions were as described in Table 1. out for 20 min. The reactions were terminated by the addition of UMP, uridine-5'-monophosphate ; UDP, uridine-5'-diphosphate; 0.1 ml 0.8 M perchloric acid. Norit-A charcoal (8 mg) was added UTP, uridine-5'-triphosphate. to each tube. After centrifugation aliquots of the supernatant a Without preincubation. were analyzed for ribose-1-phosphate (17). A blank was obtained by adding perchloric acid to a reaction mixture prior to incuba tion. Table3

%OtherShowdomycin Radioactivity recovered as to almost 100%. Unlike uridine, phosphorolysis of adenosine was not inhibited by showdomycin up to a molar ratio of added rylated (jtmoles)Oroticderivatives'014.1026.178.80l@912.04.54S62.11529.150.445.10.1320acidUMPUDPUTPphospho showdomycin to adenosine of 4. Effect of Showdomycin on Other Enzymes. UDP-glucose dehydrogenase reaction was completely inhibited by incubation of the enzyme (625 @gprotein) with 4.6 m@ano1es of showdo mycin for 10 mm at 26Â°C. Reduction of pyruvate to lactate was Effect of showdomycin on orotic acid-14C metabolism. The re not inhibited when the enzyme, lactic acid dehydrogenase (1.7 action mixtures (16 ml) contained : 40 @molesATP ; 100 @moles phosphoglyceric acid ; 28 @cmolesribose-5-phosphate ; 200 @@moles @Lg protein), was incubated with 12 @tmoles showdomycin for 10 MgCI2; 1 mmole tris(hydroxymethyl)aminomethane â€¢HC1,pH mm at 26Â°C. 7.5 ; 13 imoles orotic acid-14C (1.4 X 10@cpm) ; 1.9 or 9.1 @tmoles Reactions of Amino Acids with Showdomycin. Mixtures of showdomycin ; and 10 ml (130 mg) 100,000x g supernatant frac 12 @tmolesof showdomycin and 12 @.tmolesof glycine, histidine, tion. Incubations were carried out for 15 mm at 37Â°C.Other con or cysteine in 0.1 ml at pH 4 or 6 were incubated at 37Â°C for ditions were as described in Table 1. 1 hr. Aliquots from the incubated mixtures were analyzed by a The products were not identified. descending paper chromatography in n-butanol :water :acetic acid (4 :5 : 1 v/v/v, upper phase) . The areas corresponding to being converted predominantly to UTP. Recovery of UMP showdomycin and the amino acids were detected on the chro â€˜4Cwas increased over 200-fold at the above concentration of matograms by ultraviolet absorption and ninhydrin reaction showdomycin. When the concentration of showdomycin was respectively. The mobiities of showdomycin (R@, 0.41) and decreased to 0.12 @mole,the conversion of orotic acid to total glycine (RF, 0.19) did not change under the incubation condi phosphorylated derivatives was not inhibited. However, corn tions. With histidine a minor ninhydrin-positive material (R@, pared to the control, a significant increase in the amount of 0.09) was detected, in addition to a predominant ninhydrin UMP recovery was observed. positive area (RF, 0.16) . Cysteine (RF, 0.29) was almost quanti Effect of Showdomycin on Uridine Phosphorylase Activity. tatively converted to a new ninhydrin-positive derivative (R@, Although showdomycin was shown to be inactive as a sub 0.12). strate for uridine phosphorylase, it was a potent inhibitor of Kinetics of Inhibition Using UDP-glucose Dehydrogenase. this enzyme (Table 4) . At a 5.3 :1 molar ratio of showdomycin The results of Chart 2 show that the inhibitory effect of show to uridine, enzyme activity was inhibited 77% . This inhibitory domycin on UDP-glucose dehydrogenase is almost completely effect was not enhanced by increasing the molar ratio of in removed when showdomycin is allowed to react with cysteine hibitor to nucleoside. The effect was more pronounced when prior to the addition of enzyme. This protective effect of cys showdomycin was preincubated with the enzyme prior to the teine is abolished when it is added after preincubation of the addition of uridine. At a molar ratio of showdomycin to enzyme with the antibiotic. Methionine has no effect on show uridine of 12, preincubation increased the inhibition from 46% domycin. The data of Chart 3 show that the inhibition by

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ii â€” . â€˜V

20 __@@_0

@ I I ______I@_@ 10 20 30 40 50 Time (mm) 1/(UDPâ€”glucose)x iO@ Chart 2. Protective effect of cysteine on the inhibitory action of showdomycin with UDP-glucoae dehydrogenase. Reaction Chart 3. Inhibition of UDP-glucose dehydrogenase activity by mixtures contained, in 1 ml, 2 x 10â€”3MNAD+, 5 x 102 M tris showdomycin with respect to UDP-glucoae. Reaction mixtures, @ pH 8.0, contained, in I ml, 2 x 103 M NAD+, 5 x 102 tris . (hydroxymethyl)aminomethane-acetate, pH 8.0, 1 x 104 at UDP-glucose, and 50 units (19) of enzyme. The reactions were (hydroxymethyl)aminomethane-acetate, 35 units of enzyme, and initiated by the addition of UDP-glucose after preincubation of the indicated amount of UDP-glucose. Initial reaction velocity the mixtures for 10 or 20 min at 26Â°C.The rate of NAD+ reduc was determined in the absence (o) and presence (.) of 2.7 x tion was followed by continuous readings at 340 m@iusing a Gilford 10â€”6at showdomycin. Velocity (v) is expressed in terms of change multiple sample absorbance recorder; a, minus inhibitor (10 or 20 in A340 m@iper min. UDP, uridine-5'-diphosphate; NAD+, nice mm preincubation does not change the rate of reaction) ; b, 10 tinamide adenine dinucleotide. mm preincubation with 2.3 x 10â€”5at showdomycin (inhibitor); C, 10 mm preincubation with the inhibitor and 4.6 X 10@ at cysteine in the absence of enzyme, followed by another 10 min interconversions as the nucleoside. The resistance of the anti with enzyme ; d, 10 mm preincubation with the inhibitor in the biotic towards enzymatic phosphorolysis might be predicted presence of enzyme, followed by another 10 min with cysteine; because of the chemical stability of its carbon-linked glycosyl e, 10 mm preincubation with the inhibitor and 4.6 x 105 at bond (5) . Showdomycin is similar in this respect to pseudo methionine in the absence of enzyme, followed by another 10 min uridine, which is also not acted upon by nucleosidase (4) or with enzyme. UDP, uridine-5'-diphosphate ; NAD+, nicotinamide nucleoside phosphorylase (1). adenine dinucleotide. The decreased utilization of orotic acid produced by the higher concentration of showdomycin (Table 3) may be the result of two inhibitory effects. The major site is probably at showdomycin of the enzyme activity at pH 8.0, with respect the UMP kinase step, since the lower concentration of the to UDP-glucose, approximates uncompetitive kinetics. analog causes accumulation of UMP without limiting orotic acid conversion to nucleotides. Higher concentration of showdo DISCUSSION mycin increases the accumulation of UMP and causes a signifi cant reduction in erotic acid utilization. The latter effect may Showdomycin interferes with the metabolism of uridine by be a direct inhibition of orotidylic acid pyrophosphorylase. The inhibiting specifically UMP kinase and uridine phosphorylase inhibition, however, is not due to interference with orotidylic activities (Tables 1 and 4). These inhibitory effects are exerted acid decarboxylase, since accumulation of orotidylic acid does by the unchanged nucleoside since showdomycin is not a sub not occur. In this respect showdornycin differs from 5-hydroxy strate for nucleoside kinase or uridine phosphorylase. Thus, in uridine and 6-azauridine, which interfere with erotic acid con contrast to the majority of nucleoside analogs (6) and nucleo version to uridine nucleotide by inhibiting orotidylic acid side antibiotics (7), showdornycin inhibits specific nucleotide decarboxylase (16, 21).

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The diversity in the nature of enzymatic reactions inhibited REFERENCES by showdomycin and the marked sensitivity of the enzyme to preincubation with the antibiotic suggested the possibility that 1. Adler, M., and Gutman, A. B. Uridine Isomer (5-Ribosyluradil) the inhibitory effect may involve mechanisms which are not in Human Urine. Science, 180: 862-863, 1959. 2. Benesch, R., and Benesch, R. E. The Chemistry of the Bohr related to specific structural association with the enzyme at Effect. 1. The Reaction of N-Ethylmaleimide with the Oxygen active or allosteric sites. An alkylating action of the antibiotic linked Acid Groups of Haemoglobin. J. Biol. Chem., 288: was considered a possibility since the aglycon moiety of show 405-410, 1961. domycin is structurally related to maleimide. Maleimide de 3. Cohn, W. E. Separation of Nucleic Acid Derivatives by rivatives are known to be active alkylating agents for sulfhydryl Chromatography on Ion-Exchange Columns. In: E. Chargaff groups of amino acids, peptides, or proteins at the physiologic and J. N. Davidson (eds.), The Nucleic Acids, Vol. 1, pp. pH and temperature ranges (8, 10, 12, 13) . These compounds 211â€”241.New York: Academic Press, 1955. are also known to inactivate enzymes by a similar alkylating 4. Cohn, W. E. Pseudouridine, a Carbon-Carbon Linked Ribo nucleoside in Ribonucleic Acids : Isolation, Structure, and action (8, 13) . It has also been reported that under certain Chemical Characteristics. J. Biol. Chem., 235: 1488-1498, 1960. conditions N-substituted maleimides react with amines and 5. Darnall, K. R., Townsend, L. B., and Robins, R. K. The amino acids (20). However, the rate of reaction with sulfhydryl Structure of Showdomycin, a Novel Carbon-Linked Nucleo groups is much greater than that with amino groups. For ex side Antibiotic Related to Uridine. Proc. Natl. Acad. Sci. ample, at about pH 7 the reaction of proline with N-ethyl U. S., 57: 548-553, 1966. maleimide is only 10@ that of either cysteine or glutathione 6. Elion, G. B., and Hitchings, G. H. Metabolic Basis for the (20). Action of Analogs of Purine and Pyrimidines. In: A. Goldin, F. Hawking, and R. J. Schnitzer (eds.), Advances in Chemo Evidence that showdomycin exerts its inhibitory effect by therapy, Vol. 2, pp. 91â€”177.New York : Academic Press, 1965. alkylating action is provided by the selective effect of cysteine 7. Fox, J. J., Watanabe, K. A., and Bloch, A. Nucleoside Anti in preventing the inhibitory action of showdomycin. The data biotics. In: J. N. Davidson and W. E. Cohn (eds.), Progress of Chart 2 clearly show that preincubation of showdomycin in Nucleic Acid Research and Molecular Biology, Vol. 5, pp. with cysteine completely removes the inhibitory effect of the 281-313. New York: Academic Press, 1966. antibiotic on UDP-glucose dehydrogenase. This protective effect 8. Gregory, J. D. The Stability of N-Ethylmaleimide and its of cysteinedoesnotoccurwhentheenzymeispreincubated Reaction with Sulfhydryl Groups. J. Am. Chem. Soc., 77: 3922â€”3923,1955. with showdomycin prior to addition of cysteine. The above 9. Heinrikson, R. L., and Goldwasser, E. Studies on the Bio evidence, together with the kinetics of Chart 3 showing the synthesis of 5-Ribouracil 5'-Monophosphate in Tetrahymena uncompetitive nature of UDP-glucose dehydrogenase inhibition Pyriformis. J. Biol. Chem., 239: 1177â€”1187,1964. by showdomycin, conclusively demonstrate that the major 10. Leslie, J. Spectral Shift in the Reaction of N-Ethylmaleimide action of showdomycin is due to its alkylating effect on the with Proteins. Anal. Biochem., 10: 162â€”167,1965. enzyme. The most likely sites of alkylation are the accessible 11. Matsuura, S., Shiratori, 0., and Katagiri, K. Antitumor sulfhydryl groups. The selective inhibition of certain enzymes Activity of Showdomycin. J. Antibiotics Tokyo, Ser. A, 17: 234â€”237,1964. involved in the metabolism of pyrimidine compounds may 12. Morell, S. A., Ayers, V. E., Greenwalt, T. J., and Hoffman, P. therefore be assumed to relate to the availability of sulfhydryl Thiols of the Erythrocytes. Reaction of N-Ethylmaleimide reactive sites on the sensitive enzymes. It is of interest in this with Intact Erythrocytes. J. Biol. Chem., 289: 2696â€”2705, connection that adenosine phosphorylase is completely resistant 1964. to showdomycin whereas uridine phosphorylase is strongly 13. Mosteller, R., Ravel, J. M., and Hardesty, B. Differential inhibited. Inactivation of Soluble Reticulocyte Transfer Factors with It has been reported that maleimide derivatives are not stable N-Ethylmaleimide. Biochem. Biophys. Res. Commun., 24: at alkaline conditions (12, 20). Although most of the experi 714â€”719, 1966. 14. Nishimura, H. French Patent, M2751 September 21, 1964. ments in the present study were carried out at pH 7.4 to 8.0 Showdomycin, Extraction and Properties. Ca, 62: 2675b, for a maximum incubation time of 20 mm, it was necessary to 1965. eliminate the possibility that a small amount of degradation 15. Nishimura, H., Mayama, M., Komatsu, Y., Kato, H., product of showdomycin might be the active form of the in Shimaoka, N., and Tanaka, Y. Showdomycin, a New Antibiotic hibitor. To investigate this, showdomycin was preincubated from a Streptomyces Sp. J. Antibiotics Tokyo, Ser. A, 17: with the reaction mixture at 8.0 for 10 mm prior to the addi 148â€”155,1964. tion of the enzyme preparation and substrate (Table 2) . At 16. Pasternak, C. A., and Handschumacher, R. E. The Biochemical Activity of 6-Azauridine : Interference with Pyrimidine this condition there was no change in the inhibitory effect, mdi Metabolism in Transplantable Mouse Tumors. J. Biol. Chem., cating that showdomycin itself is directly involved in the p34: 2994â€”2997,1959. inhibition of this and other enzymes. 17. Reichard, P., and SkÃ¶ld,0. Pyrimidine Synthesis and Break down. In: S. P. Colowick and N. 0. Kaplan (eds.), Methods ACKNOWLEDGMENTS in Enzymology, Vol. 6, pp. 177â€”197.New York: Academic Press, 1963. The authors acknowledge the able technical assistance of Mrs. 18. Roy-Burman, P., Roy-Burman, S., and Visser, D. W. Utiliza Ildiko Berty and Miss Margaret A. Oberg. tion of 5,6-Dihydrouridine 5'-triphosphate in the Reaction

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Catalyzed by Eacherichia coli RNA Polymerase. Biochim. 20. Sharpless, N. E., and Flavin, M. The Reactions of Amities and Biophys. Acts, 142: 355-367, 1967. Amino Acids with Maleimides. Structure of the Reaction 19. Roy-Burman, P., Roy-Burman, S., and Visser, D. W. UDP- Products Deduced From Infrared and Nuclear Magnetic glucose Dehydrogenase. Studies with 5-Hydroxyuridine Di- Resonance Spectroscopy. Biochemistry, 5: 2963-2971, 1966. phosphate Glucose and 5,6-Dihydrouridine Dipho@phate 21. Smith, D. A., and Visser, D. W. Studies on 5-Hydroxyuridine. Glucose. J. Biol. Chem., 243: 1692â€”1697,1968. J. Biol. Chem., 240: 446-453, 1965.

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S. Roy-Burman, P. Roy-Burman and D. W. Visser

Cancer Res 1968;28:1605-1610.

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