PH-DEPENDENT INACTIVATION OF DT-DIAPHORASE BY MITOMYCIN-C AND PORFIROMYCIN

被引:0
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作者
SIEGEL, D
BEALL, H
KASAI, M
ARAI, H
GIBSON, NW
ROSS, D
机构
[1] UNIV COLORADO,HLTH SCI CTR,SCH PHARM,MOLEC TOXICOL & ENVIRONM HLTH SCI PROGRAM,BOX C238,DENVER,CO 80262
[2] UNIV COLORADO,HLTH SCI CTR,CTR CANC,DENVER,CO 80262
[3] KYOWA HAKKO KOGYO CO LTD,PHARMACEUT RES LABS,SUNTO,SHIZUOKA 411,JAPAN
[4] UNIV SO CALIF,SCH PHARM,LOS ANGELES,CA 90033
[5] UNIV SO CALIF,CTR COMPREHENS CANC,LOS ANGELES,CA 90033
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中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
Mitomycin C and porfiromycin were found to inactivate rat hepatic DT-diaphorase. Inactivation was pH dependent; little inactivation was detected at pH 5.8, but inactivation increased as the pH was raised to 7.8. Inactivation was concentration and time dependent and displayed pseudo-first-order kinetics. Inactivation was NADH dependent, indicating that reductive metabolism was necessary for inhibition. [H-3]Mitomycin C was covalently bound to DT-diaphorase during inhibition, and the stoichiometry for inactivation of DT-diaphorase by mitomycin C was approximately 0.8 nmol of mitomycin C bound/nmol of enzyme. A higher molecular mass product (60 kDa) was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of DT-diaphorase preincubated with NADH and mitomycin C at pH 7.8, suggesting that mitomycin C is capable of cross-linking DT-diaphorase. The kinetics of inhibition, requirement for NADH for inhibition, covalent binding of [H-3] mitomycin C to DT-diaphorase, and approximate 1:1 stoichiometry suggest that this inactivation process may be mechanism based. Inhibition of DT-diaphorase by mitomycin C and porfiromycin is not limited to a cell-free system and could also be observed in HT-29 cells in culture at pH 7.2. Bioactivation of mitomycin C or porfiromycin by DT-diaphorase is favored at lower pH, whereas at higher pH values enzyme alkylation and inactivation of DT-diaphorase occur. These data suggest that the success of attempts to exploit the elevated DT-diaphorase content of certain human tumors for improved chemotherapeutic response using mitomycin C or porfiromycin will depend on intracellular pH.
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页码:1128 / 1134
页数:7
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