Further characterization of a reduced nicotinamide-adenine dinucleotide phosphate-dependent aldehyde reductase from bovine brain: Inhibition by phenothiazine derivatives

Abstract

The catalytic activity of partially purified NADPH-linked aldehyde reductase (alcohol: NADP oxidoreductase, EC 1.1.1.2) from bovine brain was inhibited by certain phenothiazines. The inhibition was competitive with respect to either NADPH or aldehyde as substrate and was found to vary with the pH of the reaction mixture. At pH 7.4 the inhibitor constants ( K l values) for chlorpromazine, trifluoperazine, and thioridazine were 7.0 × 10 −4 M, 3.3 × 10 −4 M, and 8.0 × 10 −4 M respectively. Promethazine ( K l value of 45.8 × 10 −4 M) was approximately 10 times less effective than chlorpromazine in producing inhibition of enzyme activity. In addition, chlorpromazine sulfoxide did not inhibit the rate of aldehyde reduction at concentrations as high as 10 −3 M. When the K l values for the various phenothiazines were compared with the reported ability of these compounds to abolish the unconditioned avoidance response in rats, it was observed that compounds with the lowest K l values had the lowest ED 50 for abolition of this response. Studies of product inhibition of bovine brain aldehyde reductase showed that either NADP or p-nitrobenzylalcohol were competitive inhibitors with NADPH or aldehyde as the variable substrate. These observations, together with the nature of inhibition produced by the phenothiazine compounds, strongly indicate a random order of addition of substrates to the enzyme.