Molecular Pharmacology, Vol 9, 470-485, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

Reduced Diphosphopyridine Nucleotide Synergism of the Reduced Triphosphopyridine Nucleotide-Dependent Mixed-Function Oxidase System of Hepatic Microsomes

II. Role of the Type I Drug-Binding Site of Cytochrome P-450

¹ Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Studies of the DPNH synergism of the TPNH-dependent mixed-function oxidase system of hepatic microscomes using a variety of substrates revealed that synergism occurred when the substrates were type I binding compounds (aminopyrine, benzphetamine, codeine, ethylmorphine, norcodeine), but not when the substrates were type II binding compounds (aniline, p-chloro-N-methylaniline). The role of type I binding in DPNH synergism of drug metabolism was investigated by employing microsomes which varied in their abilities to produce a type I binding spectrum with ethylmorphine. This was accomplished by selecting microsomes from different animal sources (male rats of different ages, female rats, 3-methyl cholanthrene-treated rats) or by subjecting microsomes from a given source to treatments known to diminish type I binding (treatment with SKF 525-A or with phospholipase C, storage). Using ethylmorphine as the substrate, type I binding was shown to be directly correlated with DPNH synergism and with DPNH utilization. The mean ratio of DPNH utilized to ethylmorphine metabolized by the various microsomes used in the study was 1.15. No correlation was seen between DPNH utilization and rate of hydroxylation of the type II compound, aniline. No increase in DPNH utilization above that seen in the absence of substrate was observed during aniline hydroxylation. The mechanism whereby type I substrates elicit DPNH synergism is postulated to occur as follows. The first of the 2 electrons required for drug oxidation is derived from TPNH and is utilized in the reduction of the oxidized cytochrome P-450-substrate complex. The second electron is derived from either TPNH or DPNH and is transported through cytochrome b₅ to the oxygenated, reduced cytochrome P-450-substrate complex, although the possibility remains that second electrons from TPNH may be contributed by a route that circumvents cytochrome b₅. When type I substrates are introduced into the system, the rate of entry of first electrons is accelerated and second electrons from the electron pool provided by DPNH are drawn via cytochrome b₅ into the system to balance the elevated input of first electrons. When type II substrates are introduced into the system, the rate of entry of first electrons is not accelerated and there is neither the need nor the means for the system to utilize excess second electrons provided by DPNH. When only TPNH is present, the electron pool created at cytochrome b₅ is not large enough to match the pool of first electrons created by the addition of the type I substrate, and the over-all reaction is therefore slower than the DPNH-"synergized" reaction.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the able technical assistance of Miss Virginia Kickertz and Miss Viola Abbott.