Abstract
Mechanism-based inactivators serve as probes of enzyme mechanism, function, and structure. Koshland’s Reagent II (2-methoxy-5-nitrobenzyl bromide, KR-II) is a potential mechanism-based inactivator of enzymes that perform O-dealkylations. The major phenobarbital-inducible form of cytochrome P-450 in male rat liver microsomes, CYP2B1, is capable of catalyzingO-dealkylations. The interactions of KR-II with purified CYP2B1 in the reconstituted system containing P-450, NADPH:P-450 oxidoreductase, and sonicated dilaurylphosphatidyl choline were studied. The benzphetamine N-demethylase activity of CYP2B1 was inactivated by KR-II in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The inactivation also required KR-II, and the rate of activity loss was dependent on the concentration of KR-II in a saturable fashion. The inactivator concentration required for the half-maximal rate of inactivation (KI) was approximately 0.1 mM. The inactivation was not prevented by the addition of the nucleophiles dithiothreitol and glutathione, nor was it reversed by gel filtration. The present results demonstrate that KR-II is a mechanism-based inactivator of rat CYP2B1.
Footnotes
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Send reprint requests to: Dr. Paul F. Hollenberg, Department of Pharmacology, Medical Science Research Building III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail:phollen{at}umich.edu
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This research was supported in part by National Institutes of Health Grant CA16954.
- Abbreviations used are::
- P-450
- cytochrome P-450
- CYP2B1
- the major form of cytochrome P-450 from phenobarbital-pretreated rats
- KR-I
- Koshland’s Reagent I
- KR-II
- Koshland’s Reagent II
- DLPC
- dilaurylphosphatidyl choline
- Received July 22, 1998.
- Accepted February 8, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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