PT  - JOURNAL ARTICLE
AU  - D Siegel
AU  - H Beall
AU  - M Kasai
AU  - H Arai
AU  - N W Gibson
AU  - D Ross
TI  - pH-dependent inactivation of DT-diaphorase by mitomycin C and porfiromycin.
DP  - 1993 Dec 01
TA  - Molecular Pharmacology
PG  - 1128--1134
VI  - 44
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/44/6/1128.short
4100  - http://molpharm.aspetjournals.org/content/44/6/1128.full
SO  - Mol Pharmacol1993 Dec 01; 44
AB  - 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.