Further studies of the suicide inactivation of purified rat liver cytochrome P-450 by chloramphenicol.
The kinetics and reversibility of the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol have been investigated with the use of a reconstituted monooxygenase system purified from liver microsomes of phenobarbital-treated rats. At a ratio of 1 unit of NADPH-cytochrome P-450 reductase per nanomole of cytochrome P-450 and a chloramphenicol concentration of 1 mM, the t1/2 for the inactivation of cytochrome P-450 is less than 2 min. The inactivated cytochrome regains some of its activity upon incubation at 25 degrees or 37 degrees, and experiments with [14C]chloramphenicol show that this partial reactivation is accompanied by the release of some of the 14C originally bound covalently to the cytochrome P-450. Previous work has shown that the 14C-labeled material spontaneously released from 14C-labeled cytochrome P-450 is in the form of oxalic acid, and that the latter is derived from a hydroxylamine-labile adduct of chloramphenicol and cytochrome P-450 [Biochem. Pharmacol. 30:875-881 (1981)]. In the present investigation the 14C-labeled material released by hydroxylamine was identified as the hydroxamic acid of oxalic acid. Trapping experiments with the amino acid cysteine suggest that the adduct, the spontaneous degradation of which appears to be involved in the reactivation of cytochrome P-450, contains an ester rather than a thioester linkage between cytochrome P-450 and a metabolite of chloramphenicol. However, this metabolite may not be identical with chloramphenicol oxamyl chloride, which was the active metabolite implicated in the formation of the 50% covalently bound material which was stable to hydroxylamine treatment.
D000818:Animals; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D006898:Hydroxylamines / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008956:Models, Chemical; D051381:Rats; D011919:Rats, Inbred Strains
D065607:Cytochrome P-450 Enzyme Inhibitors; D006898:Hydroxylamines; D002701:Chloramphenicol