Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly ( Musca domestica)

Abstract

Components of the microsomal monooxygenase system derived from abdominal preparations of an insecticide resistant house fly ( Musca domestica, Rutgers strain) were characterized. Cytochrome P-450 isozymes with molecular weights of 49,000, 53,000, 54,000, and 58,000 were partially purified using a procedure involving chromatography on columns of phenyl Sepharose, DEAE-cellulose (DE-52), carboxymethyl-Sepharose, hydroxylapatite, and an affinity column prepared from rat liver cytochrome b 5. Activity toward a variety of pesticides, as well as other exogenous and endogenous monooxygenase substrates, was examined using intact microsomes or purified isozymes in reconstituted systems. Oxidation of benzo[ a]pyrene in microsomal preparations was high although neither O-dealkylation of 7-ethoxyresorufin nor N-dealkylation of p-chloromethylaniline could be detected. Other substrates metabolized by abdominal microsomes from Rutgers flies included testosterone, lauric acid (primarily at in-chain positions), phorate, p-nitroanisole, and aldrin. Activity of abdominal microsomes from the susceptible CSMA strain toward benzo[ a]pyrene and lauric acid was, surprisingly, higher than that from the resistant Rutgers strain. Synergism between NADPH and NADH was noted with microsomes from either strain. Reconstitution experiments were relatively unsuccessful but activity toward lauric acid was obtained using a system that included purified cytochrome P-450 fractions, purified house fly or rat liver NADPH-cytochrome P-450 reductase, an NADPH generating system, and a phospholipid. Unlike similar systems utilizing mammalian cytochrome P-450, phosphatidylethanolamine was more active than phosphatidylcholine.