[CANCER RESEARCH 28, 1106-1610,Aug@at1968]

SUMMARY

The metabolism and inhibitory effects of the carbon-linkednucleoside antibiotic showdomycin were investigated in cellfree preparatiozis of Ehrlich ascites cells. The antibiotic is nota substrate for nucleoside kinase or nucleoside phosphorylase.Showdomycin shows inhibitory effects on certain enzymes in anEhrlich ascites cell preparation, which are involved in uridineand orotic acid metabolism. It inhibits üridine-5'-monophosphokinase, uridine phosphorylase, and possibly orotidylic acidpyrophosphorylase reactions, but has no effect on the activityof uridine kinase or adenosine phosphorylase. Showdomycinstrongly inhibits bovine liver uridine-5'-diphosphate.@cr.D-glucosedehydrogenase but has no effect on rabbit muscle lactic aciddehydrogenase. The selective inhibition of certain enzymes byshowdomycin may be related to the alkylating property of itsmaleimide structure, which is known to specifically react withsulfhydryl groups.

INTRODUCflON

The antibiotic, showdomycin, first isolated by Nishimuraet at. (15) from Streptomyces showdoensis, inhibits growth ofseveral Gram-positive and Gram-negative bacteria and is particularly active against Streptococcus haemotyticus (15) and

1 This investigation was supported by research grants from

USPHS (CA 02373-12), the American Cancer Society (E-412)and (in part) by a Dernham Senior Fellowship of the AmericanCancer Society, California Division (No. D120).

Received January 26, 1968; accepted April 21, 1968.

HOC@

OH OH

Streptococcus pyogenes (14). Showdomycin has antitumoractivity against Ehrlich ascites cells both in vitro and in vivoand is active against cultured HeLa cells ( 11, 14, 15) . Itsstructure has been established as 3-$-o-ribofuranosylmaleimide(5), a carbon-linked nucleoside antibiotic.

The structural similarity of showdomycin, uridine, andpseudouridine are apparent from the formulae (Chart 1).Darnall et at. (5) have pointed out that showdomycin may beviewed as pseudouridine which has lost an -NH group in thecontraction to a five-membered ring.

The unique structure of showdomycin, its structural similarity to uridine, and its biologic properties prompted this investigation of its metabolism and its effect on various enzymes,particularly those involved in uridine metabolism.

MATERIAL AND METHODS

Showdomycin was obtained as a gift from Dr. Ronald K.Robins of the University of Utah, Salt Lake City, Utah, andDr. Haruo Nishimura of Shionogi Research Laboratory,Fukushima-Ku, Osaka, Japan. The purity of the compoundwas determined in two descending paper chromatographicsystems. The compound migrated as a single ultraviolet absorbing area in ethanol 0.5 M ammonium acetate, pH 6.5 (5:2v/v) and isopropanol: concentrated HC1: 1120 (65:16.7:18.3v/v/v) solvent systems, the respective RF values being 0.64and 0.68. Uridine-2-14C and UMP-2-'4C2 were purchased fromSchwarz BioResearch, Inc. Orotic acid-6-14C was obtainedfrom New England Nuclear Corporation. Nucleoside, nucleotides, ribose-5'-phosphate, and 3-phospho-D-glyceric acid(sodium salt) were obtained from California Corporation forBiochemical Research.

A supernatant fraction (100,000 x g) from a hyperdiploidstrain of Ehrlich ascites tumor cells was prepared as describedpreviously (18, 21). A crude preparation of uridine phosphorylase was obtained from Ehrlich ascites cells in the followingmanner. Ascites cells were washed as described previously(21) and disrupted using a French press at 1800 lb/sq inch at0-5°C in 2 volumes of buffer containing 0.05 M potassiumphosphate, pH 7.4, and 0.13 M potassium chloride. An additional amount of buffer was added to the disrupted cells to make

2 Abbreviations used are : UMP, uridine-5'-monophosphate;

UDP, uridine-5'-diphosphate ; UDP-glucose, uridlne-5'-diphoephate-a-n-glucose ; UTP, uridine-S'-triphosphate ; Tris, tria(hydroxymethyl)aminomethane ; NAD+, nicotinamide adenine dinucleotide.

SHOWDOMYCIN URIDINE

Chart 1. Graphic formulas.

PSEUDOURIDINE

AUGUST 1968 1605

Showdomycin, A New Nucleoside Antibiotic'

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

HN

0 _.-@

HOCH2 0

OH OH

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Showdoinycinadded

(@unoles)Radioactivity

recovered as%UMPUDP

sugarUDPUTPTotal

phosphorylated

derivatives00.5088.670.180.05.029.305.050.084.310.063.302.720.686.6

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

a final concentration of 3 ml of buffer per gm of cells. Thesuspension was centrifuged for 60 min at 85,000 x g. Thesupernatant enzyme preparation contained 12-14 mg protein/ml and had a specific activity of 110-160 (17). This preparation was used for assay of uridine phosphorylase and adenosinephosphorylase.

Preparation of acid-soluble fractions from the various incubation mixtures and separation of phosphorylated derivativesby chromatography on Dowex 1 (formate) were performedas described previously (18).

Uridine diphosphate glucose dehydrogenase was purchasedfrom Sigma Chemical Company and was assayed (19) byfollowing NAD+ reduction. Rabbit muscle lactic dehydrogenasewas obtained from California Corporation for BiochemicalResearch and was assayed by determining the rate of oxidationof reduced NM) with pyruvate as substrate.

RESULTS

Effect of Kinases on Showdomycin. Since inhibitory effectsof most nucleoside derivatives are dependent upon a priorconversion to phosphorylated derivatives, it was of interest todetermine the susceptibility of showdomycin to kinase activities. Showdomycin was incubated at pH 7.8, 7.0, and 6.2 witha supernatant fraction prepared from Ehrlich ascites cellshaving high levels of uridine, UMP, and UDP kinase activity(18, 21) . Incubation mixtures were treated as described previously (18) except that the separation of acid-soluble cornponents was carried out on a Dowex 1 (chloride) columnusing a gradient concentration of hydrochloric acid and sodiumchloride. Initially the mixer contained 1 liter of water and thereservoir contained 0.01 N hydrochloric acid. After collectionof about 1 liter of eluent, the solution in the mixer was changedto 1 liter of 0.01 N hydrochloric acid and that in the reservoirto 0.2 M NaC1. After collection of about 2 additional liters ofeluent, the column was eluted directly with 0.5 M NaCl in 0.01N hydrochloric acid. Since showdomycin in acidic solution has

a characteristically high absorbance at 220@ and a very lowabsorbance at 260 m@ ( 15) , the absorbance of the effluent wasmeasured at 220 m@ and compared with a control which washandled in an identical manner, except that showdomycin was

omitted from the incubation mixture. The detection limit ofthe photometric assay procedure is about 10 optical densityunits which represents about 1% of the added showdomycin.The fractions were also tested with Elson-Morgan's color reagent, which gives a red color with showdomycin (15). Theresults, with and without showdornycin, were identical basedon these assay procedures, showing that showdomycin is not.converted to phosphorylated derivatives by enzyme preparations which converted 50-70% of uridine-'4C to UTP.

Effect of Showdomycin on Uridine Metabolism. The resultsof experiments designed to determine the effect of showdomycin on the conversion of uridine-'4C to phosphorylated derivatives are shown in Table 1. The data show that showdomycindid not inhibit uridine kinase but strongly inhibited the conversion of UMP to UDP. The latter effect increased with increasing concentrations of showdomycin. In the presence of2.5 @molesshowdomycin per ml reaction mixture, UMP-14Crepresented more than 70% of the radioactive nucleotides,whereas in the absence of showdornycin, about 90% of theacid-soluble nucleotides was recovered as UTP. Uridine kinaseactivity was not inhibited by showdomycin under the sameconditions.

Since the previous experiments indicated that showdornycinspecifically inhibited UMP kinase, the effect of the analog onthe phosphorylation of UMP-14C was investigated. In thepresence of 1.6 @molesshowdomycin per ml reaction mixture,UTP formation was inhibited about 50% (Table 2). Whenthe enzyme mixture was preincubated with the same concentration of showdomycin prior to the addition of UMP-14C,UMP kinase activity was completely inhibited, whereas 94%of UMP was converted to the triphosphate in the absence ofshowdomycin. Preincubation of showdomycin with the reactionmixture prior to the addition of both UMP-'4C and enzyme didnot increase the inhibitory effect.

Effect of Showdomycin on the Conversion of Orotic Acidto Uridine Nucleotides. Showdomycin inhibited the conversionof orotic acid-'4C to uridine nucleotides (Table 3) . At a concentration of 0.56 @&moleshowdomycin per ml reaction mixture,about 50% of the total radioactivity was recovered as eroticacid, whereas in the absence of showdomycin, less than 15%of the radioactivity remained as orotic acid, the remainder

Table 1

Effect of showdomycin on uridine-14C phosphorylation. The reaction mixtures (4 ml) contamed : 20 @molesATP ; 10 @@molesphosphoglyceric acid ; 50 @@molesMgCl2 ; 250 @&molestris(hydroxymethyl)aminomethane-HC1, pH 7.5; 1 @moleuridine-14C (7.6 x 105 cpm) ; 5 or10 @imolesshowdomycin ; and 22 ml (29 mg) 100,000 x g supernatant fraction. Incubationswere carried out for 10 mm at 37°Cand terminated with 1.0 ml 4.0 M perchloric acid. Theradioactive 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.

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Showdomycinadded (i@moIes)Addition

afterpreincubationRadiosctivity

recoveredas%UMPTJDPUTP0UMP-'@C0394.6593.905aNone

UMP-'@C,41.7410.0946.425enzyme40.2810.6747.675UMP@14C97.730010TJMP@14C98.7000

Addition (jimoles)Ribose-1-phosphateformed(manoles)Without

preincubationPreincubstionUridineShowdomycin1.0031.02012202.71.00.6192.05921.012107.90.91.01.982.00.91.02271.01.05.346.01.010.053.0

Radioactivity recovered as%OtherShowdomycin

added(jtmoles)Orotic acidUMPUDPUTPphospho

rylatedderivatives'014.1026.178.80l@912.04.54S62.11529.150.445.10.1320

Showdomycin

Table 2 Table 4

Effect of showdomycin on UMP@14C phosphorylation. The reaction mixture (3 ml) contained : 7 @molesATP ; 20 jimoles phosphoglyceric acid ; 33 @molesMgCl2 ; 250 @molestris(hydroxymethyl)aminomethane •HC1, pH 8.0; 1 @moleUMP_14C (1 x 10@cpm) ; 5 or 10 @molesshowdomycin; and 1 ml (13 mg) 100,000 xg supernatant fraction. The reaction mixtures were incubated for10 mm at 37°Cin the absence of UMP-'@C or both UMP@14Candenzyme as indicated and after their addition incubation was continued for an additional 10 min. In experiments where preincubation was not carried out, UMP@14C and the enzyme were addedto the reaction mixture at zero time and incubations were carriedout for 10 mm. Other conditions were as described in Table 1.UMP, uridine-5'-monophosphate ; UDP, uridine-5'-diphosphate;UTP, uridine-5'-triphosphate.

a Without preincubation.

Effect of showdomycin on uridine phosphorylase activity. Thereaction mixtures (0.15 ml) contained : 15 @@molespotassium phosphate buffer, pH 7.4; 0.1 ml (1.4 mg) enzyme preparation; andthe indicated amounts of showdomycin. After preincubation ofthe reaction mixtures for 10 min at 37°C,1 @&moleuridine wasadded and the incubation was continued for 20 mm. In experiments where preincubation was not carried out, uridine was addedto the reaction mixture at zero time and incubations were carriedout for 20 min. The reactions were terminated by the addition of0.1 ml 0.8 M perchloric acid. Norit-A charcoal (8 mg) was addedto each tube. After centrifugation aliquots of the supernatantwere analyzed for ribose-1-phosphate (17). A blank was obtainedby adding perchloric acid to a reaction mixture prior to incubation.

to almost 100%. Unlike uridine, phosphorolysis of adenosinewas not inhibited by showdomycin up to a molar ratio ofshowdomycin to adenosine of 4.

Effect of Showdomycin on Other Enzymes. UDP-glucosedehydrogenase reaction was completely inhibited by incubationof the enzyme (625 @gprotein) with 4.6 m@ano1es of showdomycin for 10 mm at 26°C. Reduction of pyruvate to lactate wasnot inhibited when the enzyme, lactic acid dehydrogenase (1.7@Lg protein), was incubated with 12 @tmoles showdomycin for 10

mm at 26°C.Reactions of Amino Acids with Showdomycin. Mixtures of

12 @tmolesof showdomycin and 12 @.tmolesof glycine, histidine,or cysteine in 0.1 ml at pH 4 or 6 were incubated at 37°C for1 hr. Aliquots from the incubated mixtures were analyzed bydescending paper chromatography in n-butanol :water :aceticacid (4 :5 : 1 v/v/v, upper phase) . The areas corresponding toshowdomycin and the amino acids were detected on the chromatograms by ultraviolet absorption and ninhydrin reactionrespectively. The mobiities of showdomycin (R@, 0.41) andglycine (RF, 0.19) did not change under the incubation conditions. With histidine a minor ninhydrin-positive material (R@,0.09) was detected, in addition to a predominant ninhydrinpositive area (RF, 0.16) . Cysteine (RF, 0.29) was almost quantitatively converted to a new ninhydrin-positive derivative (R@,0.12).

Kinetics of Inhibition Using UDP-glucose Dehydrogenase.The results of Chart 2 show that the inhibitory effect of showdomycin on UDP-glucose dehydrogenase is almost completelyremoved when showdomycin is allowed to react with cysteineprior to the addition of enzyme. This protective effect of cysteine is abolished when it is added after preincubation of theenzyme with the antibiotic. Methionine has no effect on showdomycin. The data of Chart 3 show that the inhibition by

Table3

Effect of showdomycin on orotic acid-14C metabolism. The reaction mixtures (16 ml) contained : 40 @molesATP ; 100 @molesphosphoglyceric acid ; 28 @cmolesribose-5-phosphate ; 200 @@molesMgCI2; 1 mmole tris(hydroxymethyl)aminomethane •HC1, pH7.5 ; 13 imoles orotic acid-14C (1.4 X 10@cpm) ; 1.9 or 9.1 @tmolesshowdomycin ; and 10 ml (130 mg) 100,000x g supernatant fraction. Incubations were carried out for 15 mm at 37°C.Other conditions were as described in Table 1.

a The products were not identified.

being converted predominantly to UTP. Recovery of UMP‘4Cwas increased over 200-fold at the above concentration ofshowdomycin. When the concentration of showdomycin wasdecreased to 0.12 @mole,the conversion of orotic acid to totalphosphorylated derivatives was not inhibited. However, cornpared to the control, a significant increase in the amount ofUMP recovery was observed.

Effect of Showdomycin on Uridine Phosphorylase Activity.Although showdomycin was shown to be inactive as a substrate for uridine phosphorylase, it was a potent inhibitor ofthis enzyme (Table 4) . At a 5.3 :1 molar ratio of showdomycinto uridine, enzyme activity was inhibited 77% . This inhibitoryeffect was not enhanced by increasing the molar ratio of inhibitor to nucleoside. The effect was more pronounced whenshowdomycin was preincubated with the enzyme prior to theaddition of uridine. At a molar ratio of showdomycin touridine of 12, preincubation increased the inhibition from 46%

AUGUST 1968 1607

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

40

30

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20

10

Time (mm)

10 20 30 40 50

1/(UDP—glucose)x iO@

Chart 3. Inhibition of UDP-glucose dehydrogenase activity byshowdomycin 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, 35 units of enzyme, andthe indicated amount of UDP-glucose. Initial reaction velocitywas determined in the absence (o) and presence (.) of 2.7 x10—6at showdomycin. Velocity (v) is expressed in terms of changein A340 m@iper min. UDP, uridine-5'-diphosphate; NAD+, nicetinamide adenine dinucleotide.

interconversions as the nucleoside. The resistance of the antibiotic towards enzymatic phosphorolysis might be predictedbecause of the chemical stability of its carbon-linked glycosylbond (5) . Showdomycin is similar in this respect to pseudouridine, which is also not acted upon by nucleosidase (4) ornucleoside phosphorylase (1).

The decreased utilization of orotic acid produced by thehigher concentration of showdomycin (Table 3) may be theresult of two inhibitory effects. The major site is probably atthe UMP kinase step, since the lower concentration of theanalog causes accumulation of UMP without limiting oroticacid conversion to nucleotides. Higher concentration of showdomycin increases the accumulation of UMP and causes a significant reduction in erotic acid utilization. The latter effect maybe a direct inhibition of orotidylic acid pyrophosphorylase. Theinhibition, however, is not due to interference with orotidylicacid decarboxylase, since accumulation of orotidylic acid doesnot occur. In this respect showdornycin differs from 5-hydroxyuridine and 6-azauridine, which interfere with erotic acid conversion to uridine nucleotide by inhibiting orotidylic aciddecarboxylase (16, 21).

Chart 2. Protective effect of cysteine on the inhibitory actionof showdomycin with UDP-glucoae dehydrogenase. Reactionmixtures contained, in 1 ml, 2 x 10—3M NAD+, 5 x 102 M tris

. (hydroxymethyl)aminomethane-acetate, pH 8.0, 1 x 104 at

UDP-glucose, and 50 units (19) of enzyme. The reactions wereinitiated by the addition of UDP-glucose after preincubation ofthe mixtures for 10 or 20 min at 26°C.The rate of NAD+ reduction was followed by continuous readings at 340 m@iusing a Gilfordmultiple sample absorbance recorder; a, minus inhibitor (10 or 20mm preincubation does not change the rate of reaction) ; b, 10mm preincubation with 2.3 x 10—5at showdomycin (inhibitor);C, 10 mm preincubation with the inhibitor and 4.6 X 10@ atcysteine in the absence of enzyme, followed by another 10 minwith enzyme ; d, 10 mm preincubation with the inhibitor in thepresence of enzyme, followed by another 10 min with cysteine;e, 10 mm preincubation with the inhibitor and 4.6 x 105 atmethionine in the absence of enzyme, followed by another 10 minwith enzyme. UDP, uridine-5'-diphosphate ; NAD+, nicotinamideadenine dinucleotide.

showdomycin of the enzyme activity at pH 8.0, with respectto UDP-glucose, approximates uncompetitive kinetics.

DISCUSSION

Showdomycin interferes with the metabolism of uridine byinhibiting specifically UMP kinase and uridine phosphorylaseactivities (Tables 1 and 4). These inhibitory effects are exertedby the unchanged nucleoside since showdomycin is not a substrate for nucleoside kinase or uridine phosphorylase. Thus, incontrast to the majority of nucleoside analogs (6) and nucleoside antibiotics (7), showdornycin inhibits specific nucleotide

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Showdomycin

The diversity in the nature of enzymatic reactions inhibitedby showdomycin and the marked sensitivity of the enzyme topreincubation with the antibiotic suggested the possibility thatthe inhibitory effect may involve mechanisms which are notrelated to specific structural association with the enzyme atactive or allosteric sites. An alkylating action of the antibioticwas considered a possibility since the aglycon moiety of showdomycin is structurally related to maleimide. Maleimide derivatives are known to be active alkylating agents for sulfhydrylgroups of amino acids, peptides, or proteins at the physiologicpH and temperature ranges (8, 10, 12, 13) . These compoundsare also known to inactivate enzymes by a similar alkylatingaction (8, 13) . It has also been reported that under certainconditions N-substituted maleimides react with amines andamino acids (20). However, the rate of reaction with sulfhydrylgroups is much greater than that with amino groups. For example, at about pH 7 the reaction of proline with N-ethylmaleimide is only 10@ that of either cysteine or glutathione(20).

Evidence that showdomycin exerts its inhibitory effect byalkylating action is provided by the selective effect of cysteinein preventing the inhibitory action of showdomycin. The dataof Chart 2 clearly show that preincubation of showdomycin

with cysteine completely removes the inhibitory effect of theantibiotic on UDP-glucose dehydrogenase. This protective effectof cysteinedoesnotoccurwhentheenzymeis preincubatedwith showdomycin prior to addition of cysteine. The aboveevidence, together with the kinetics of Chart 3 showing theuncompetitive nature of UDP-glucose dehydrogenase inhibitionby showdomycin, conclusively demonstrate that the majoraction of showdomycin is due to its alkylating effect on theenzyme. The most likely sites of alkylation are the accessiblesulfhydryl groups. The selective inhibition of certain enzymesinvolved in the metabolism of pyrimidine compounds maytherefore be assumed to relate to the availability of sulfhydrylreactive sites on the sensitive enzymes. It is of interest in thisconnection that adenosine phosphorylase is completely resistantto showdomycin whereas uridine phosphorylase is stronglyinhibited.

It has been reported that maleimide derivatives are not stableat alkaline conditions (12, 20). Although most of the experiments in the present study were carried out at pH 7.4 to 8.0for a maximum incubation time of 20 mm, it was necessary toeliminate the possibility that a small amount of degradationproduct of showdomycin might be the active form of the inhibitor. To investigate this, showdomycin was preincubatedwith the reaction mixture at 8.0 for 10 mm prior to the addition of the enzyme preparation and substrate (Table 2) . Atthis condition there was no change in the inhibitory effect, mdicating that showdomycin itself is directly involved in theinhibition of this and other enzymes.

ACKNOWLEDGMENTS

The authors acknowledge the able technical assistance of Mrs.Ildiko Berty and Miss Margaret A. Oberg.

REFERENCES

1. Adler, M., and Gutman, A. B. Uridine Isomer (5-Ribosyluradil)in Human Urine. Science, 180: 862-863, 1959.

2. Benesch, R., and Benesch, R. E. The Chemistry of the BohrEffect. 1. The Reaction of N-Ethylmaleimide with the Oxygenlinked Acid Groups of Haemoglobin. J. Biol. Chem., 288:405-410, 1961.

3. Cohn, W. E. Separation of Nucleic Acid Derivatives byChromatography on Ion-Exchange Columns. In: E. Chargaffand J. N. Davidson (eds.), The Nucleic Acids, Vol. 1, pp.211—241.New York: Academic Press, 1955.

4. Cohn, W. E. Pseudouridine, a Carbon-Carbon Linked Ribonucleoside in Ribonucleic Acids : Isolation, Structure, andChemical Characteristics. J. Biol. Chem., 235: 1488-1498, 1960.

5. Darnall, K. R., Townsend, L. B., and Robins, R. K. TheStructure of Showdomycin, a Novel Carbon-Linked Nucleoside Antibiotic Related to Uridine. Proc. Natl. Acad. Sci.U. S., 57: 548-553, 1966.

6. Elion, G. B., and Hitchings, G. H. Metabolic Basis for theAction of Analogs of Purine and Pyrimidines. In: A. Goldin,F. Hawking, and R. J. Schnitzer (eds.), Advances in Chemotherapy, Vol. 2, pp. 91—177.New York : Academic Press, 1965.

7. Fox, J. J., Watanabe, K. A., and Bloch, A. Nucleoside Antibiotics. In: J. N. Davidson and W. E. Cohn (eds.), Progressin Nucleic Acid Research and Molecular Biology, Vol. 5, pp.281-313. New York: Academic Press, 1966.

8. Gregory, J. D. The Stability of N-Ethylmaleimide and itsReaction with Sulfhydryl Groups. J. Am. Chem. Soc., 77:3922—3923,1955.

9. Heinrikson, R. L., and Goldwasser, E. Studies on the Biosynthesis of 5-Ribouracil 5'-Monophosphate in TetrahymenaPyriformis. J. Biol. Chem., 239: 1177—1187,1964.

10. Leslie, J. Spectral Shift in the Reaction of N-Ethylmaleimidewith Proteins. Anal. Biochem., 10: 162—167,1965.

11. Matsuura, S., Shiratori, 0., and Katagiri, K. AntitumorActivity of Showdomycin. J. Antibiotics Tokyo, Ser. A, 17:234—237,1964.

12. Morell, S. A., Ayers, V. E., Greenwalt, T. J., and Hoffman, P.Thiols of the Erythrocytes. Reaction of N-Ethylmaleimidewith Intact Erythrocytes. J. Biol. Chem., 289: 2696—2705,1964.

13. Mosteller, R., Ravel, J. M., and Hardesty, B. DifferentialInactivation of Soluble Reticulocyte Transfer Factors withN-Ethylmaleimide. Biochem. Biophys. Res. Commun., 24:714—719, 1966.

14. Nishimura, H. French Patent, M2751 September 21, 1964.Showdomycin, Extraction and Properties. Ca, 62: 2675b,1965.

15. Nishimura, H., Mayama, M., Komatsu, Y., Kato, H.,Shimaoka, N., and Tanaka, Y. Showdomycin, a New Antibioticfrom a Streptomyces Sp. J. Antibiotics Tokyo, Ser. A, 17:148—155,1964.

16. Pasternak, C. A., and Handschumacher, R. E. The BiochemicalActivity of 6-Azauridine : Interference with PyrimidineMetabolism in Transplantable Mouse Tumors. J. Biol. Chem.,p34: 2994—2997,1959.

17. Reichard, P., and Sköld,0. Pyrimidine Synthesis and Breakdown. In: S. P. Colowick and N. 0. Kaplan (eds.), Methodsin Enzymology, Vol. 6, pp. 177—197.New York: AcademicPress, 1963.

18. Roy-Burman, P., Roy-Burman, S., and Visser, D. W. Utilization of 5,6-Dihydrouridine 5'-triphosphate in the Reaction

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S. Roy-Burman, P. Roy-Burman, and D. W. Vi@sser

Catalyzed by Eacherichia coli RNA Polymerase. Biochim. 20. Sharpless, N. E., and Flavin, M. The Reactions of Amities andBiophys. 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 Magneticglucose 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: 1692—1697,1968. J. Biol. Chem., 240: 446-453, 1965.

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