Molecular Pharmacology, Vol 10, 1004-1008, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Discrepancy in the Measurement of Reduced Triphosphopyridine Nucleotide Oxidized during Ethylmorphine N-Demethylation Due to the Presence of a Nucleotide Pyrophosphatase

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

The failure to establish the required 1:1 stoichiometry for TPNH utilization and product formation for the hepatic drug-metabolizing system involving cytochrome P-450 has been attributed to TPNH utilization by endogenous substrates. Attempts to achieve stoichiometry by subtracting endogenous TPNH utilization from TPNH utilization in the presence of substrate have not proved satisfactory. When the disappearance of TPNH was monitored both enzymatically and by observing the decline in spectral absorbance at 340 nm in the presence and absence of the substrate, ethylmorphine, values were identical initially, but became increasingly divergent with incubation time. Less of the apparent TPNH was present when the enzymatic procedure was used. The high values obtained by the spectrophotometric method were shown to be due to reduced nicotinamide mononucleotide, which also gives a maximum absorption peak at 340 nm. NMNH is formed through the action of microsomal nucleotide pyrophosphatase. Moreover, ethylmorphine inhibits NMNH formation by competing for TPNH for its N-demethylation. A 1:1 stoichiometry of TPNH utilization to formaldehyde formation was possible when calculations were corrected to include rates of NMNH formation. EDTA (0.2 mM) inhibited NMNH formation and thereby enabled the calculation of a 1:1 stoichiometric relationship. ATP and 5'-AMP also inhibited nucleotide pyrophosphatase activity.