The effects of sulphydryl reagents on the binding and mixed function oxidation of hexobarbital in rat hepatic microsomes.
1. The effects of the sulphydryl reagents p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide on the binding spectrum, oxygen consumption and formation of a suspected substrate-cytochrome P-450-oxygen complex for hexobarbital in rat liver microsomes were investigated. 2. The oxygen consumption caused by hexobarbital oxidation was inhibited non-competitively by all three agents, with 50% inhibition at 4 times 10(-5) M for p-chloromercuribenzoate, 3-7 times 10(-4) M for N-ethylmaleimide and 1-9 times 10(-3) M for iodoacetamide. Cysteamine protected and at least partially reversed this inhibition. 3. p-chloromercuribenzoate inhibited the formation of the cytochrome P-450-substrate-oxygen complex, while N-ethylmaleimide and iodoacetamide also inhibited the formation of this complex but to a lesser extent. The p-chloromercuribenzoate inhibition was protected against and reversed by cysteamine. 4. p-Chloromercuribenzoate and N-ethylmaleimide caused a 50% reduction in the magnitude of the hexobarbital-induced binding spectrum, and this was paralleled by the conversion of cytochrome P-450 to cytochrome P-420. Cysteamine protected against this effect but could not reverse it. Iodoacetamide had no effect on the binding spectrum of hexobarbital and failed to convert cytochrome P-450 to cytochrome P-420. 5. Points of attack within the reaction sequence of drug oxidation are tentatively ascribed to the sulphydryl reagents used in this study.
D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003543:Cysteamine / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006591:Hexobarbital / Q000378:metabolism*; D007460:Iodoacetamide / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002729:Chloromercuribenzoates; D013439:Sulfhydryl Reagents; D009249:NADP; D003543:Cysteamine; D003577:Cytochrome P-450 Enzyme System; D006591:Hexobarbital; D005033:Ethylmaleimide; D007460:Iodoacetamide