pH-dependent inactivation of DT-diaphorase by mitomycin C and porfiromycin.
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. [3H]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 [3H] 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.
Siegel
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D
Molecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262.
Beall
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H
Kasai
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M
Arai
H
H
Gibson
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NW
Ross
D
D
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016685:Mitomycin / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors*; D011160:Porfiromycin / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured
D009243:NAD; D016685:Mitomycin; D016660:NAD(P)H Dehydrogenase (Quinone); D011160:Porfiromycin