Molecular Pharmacology, Vol 15, 396-409, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Mechanism of the Nucleotide Pyrophosphatase Induced Distortion of Stoichiometry of TPNH Utilization and Product Formation by Hepatic Cytochrome P-450 Linked N-Demethylase Systems

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

By definition, an hepatic cytochrome P-450-and TPNH-linked monooxygenase reaction requires that a mole of TPNH be utilized for each mole of product formed. When rat hepatic microsomes have been used to study the stoichiometry of N-demethylase reactions, considerably more TPNH utilization has usually been observed than can be accounted for by product (HCHO) formation. The presence of nucleotide pyrophosphatase in microsomes has been shown to cause the apparent substrate-induced enhancement of endogenous TPNH oxidation. Demethylation of the substrate (aminopyrine, benzphetamine, codeine or ethylmorphine) increases the rate of TPNH oxidation and this enhances the rate limitation of TPNH for the pyrophosphatase reaction (a first order reaction) without inflicting a TPNH rate limitation on the endogenous oxidation of TPNH (a zero order reaction). As a consequence, tess 2'5' ADP—an inhibitor of TPNH oxidation—is formed from TPNH by the pyrophosphatase when the substrate is present than when it is absent. Because less 2'5' ADP is formed in the presence of substrate, less inhibition of endogenous TPNH oxidation occurs; substrate thus appears to stimulate endogenous TPNH oxidation when in fact it is only permitting the reaction to proceed at a less inhibited rate. When EDTA (0.2 mM) or 5' AMP (1 mM) is used to inhibit pyrophosphatase activity, a stoichiometry of TPNH oxidized:HCHO formed of unity was observed when microsomes from several mammalian species were used. Unity was also observed when the influence of pyrophosphatase was diminished by increasing the ratio of demethylase activity to pyrophosphatase activity in microsomes by administering phenobarbital to rats; by modulating the ratios of the two enzymes in this manner, it was shown that disparity in the 1:1 ratio of TPNH oxidation: HCHO formation is directly related to the nucleotide pyrophosphatase content of the microsomes. When EDTA or 5' AMP is added to the medium, the rate of monooxygenase activity of hepatic microsomes can be measured by monitoring the disappearance of the absorbance of TPNH at 340 nm.