Aldrin Epoxidation Catalyzed by Purified Rat‐Liver Cytochromes P‐450 and P‐448 High Selectivity for Cytochrome P‐450

Abstract

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased 3-fold by addition of dilauroylphosphatidylcholine and was further stimulated 2-fold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 .+-. 2 .mu.M. SKF 525-A, at a concentration of 250 .mu.M, inhibited aldrin epoxidation by 65%; 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 .mu.M. Addition of ethanol markedly increased epoxidase activity. The increase was 3-fold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was < 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 .+-. 7 .mu.M. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 [50% inhibition] was 0.05, 0.2 and 800 mM, respectively. Aldrin is apparently a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The 2 forms of aldrin epoxidase can be characterized by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.