Microsomal cytochrome P-450 from the housefly, Musca domestica: Assay and spectral characterization

Abstract

The effects of assay conditions on spectral characterization of microsomal cytochrome P-450 from insecticide-resistant (Fc) and insecticide-susceptible (CSMA) strains of the housefly, ( Musca domestica) were investigated. The specific activity of cytochrome P-450 was higher in microsomes prepared from isolated abdomens than in those from whole flies. Buffers of low molarity (<0·1 M) caused denaturation of cytochrome P-450 and the optimum pH values, in terms of cytochrome P-450 content, were pH 7·0 and 7·2 for microsomes of Fc and CSMA houseflies respectively. Guidelines were established to reduce errors in measurement and interpretation of substrate difference spectra. Cytochrome P-420 did not exhibit normal type I and type II difference spectra, however, its presence in microsomes interfered with the evaluation of the type III difference spectra of ethyl isocyanide and pyridine. Some strain specific differences were noted in the oxidized difference spectra of type I and type II ligands and in those obtained with microsomes pretreated with either NADH or NADPH. Microsomes from neither strain exhibited normal type I difference spectra with benzphetamine in the presence of dithionite. The data obtained are discussed in relation to current methodology used with microsomal cytochrome P-450 from insects and other taxonomic groups.