Molecular Pharmacology, Vol 9, 455-469, 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

I. Effects of Activation and Inhibition of the Fatty Acyl Coenzyme A Desaturation System

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

Time first of the 2 electrons required for the N-demethylation of ethylmorphine by the microsomal mixed-function oxidase system of liver is thought to be donated by TPNH, whereas the second has been postulated to be derivable from either TPNH or DPNH and transported to the cytochrome P-450 system via cytochrome b₅. DPNH is believed to support a larger pool of second electrons at cytochrome b₅ than can be supplied by TPNH, and this would explain the synergistic effect of DPNH on TPNH-dependent mixed-function oxidase reactions. This concept was strengthened by the current studies, which altered the pool of second electrons at cytochrome b₅ by manipulating the shunt of electrons from cytochrome b₅ to the microsomal fatty acyl-CoA desaturation system. When the activity of this shunt was inhibited with cyanide, DPNH synergism of ethylmorphine N-demethylation was increased from 30% (no cyanide) to 90% (0.1 or 0.5 mM cyanide). Cyanide had little or no effect on N-demethylation when TPNH was the sole source of electrons. When the activity of the desaturation system was stimulated by the addition of stearoyl-CoA, a natural substrate for the system, rates of N-demethylation declined when TPNH was the sole donor of electrons and when both TPNH and DPNH were present. Cyanide reversed the inhibitory effect of stearoyl-CoA. These results are interpreted to mean that second electrons, which are normally transported from DPNH through cytochrome b₅ to the cytochrome P-450 system, are dissipated in large part via cyanide-sensitive systems engaged in the oxidation of endogenous substrates; cyanide largely prevents this dissipation of second electrons, more of the electron pool at cytochrome b₅ is made available to the cytochrome P-450 system, and DPNH synergism is enhanced. Cyanide is without effect on the cytochrome P-450 system when TPNH is the sole source of electrons, because the steady-state concentration of reduced cytochrome b₅ that can be maintained by TPNH is below the level where a serious loss of electrons to endogenous systems via cytochrome b₅ can occur. Stearoyl-CoA increases the dissipation of electrons by serving as a substrate for the cyanide-sensitive desaturation system. It is postulated that stearoyl-CoA inhibits ethylmorphine N-demethylation because the desaturation system and the mixed-function oxidase system compete for electrons supplied by an unidentified electron transfer component, which accepts electrons from either DPNH or TPNH via cytochrome b₅. These concepts were reinforced by studies in which rates of DPNH utilization were correlated with rates of ethylmorphine N-demethylation.

Note:
ACKNOWLEDGMENT The authors gratefully acknowledge the able technical assistance of Miss Virginia Kickertz.

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