Characterization of antibodies to house fly NADPH-cytochrome [formula omitted] reductase

Abstract

Antibodies to solubilized and purified NADPH-cytochrome P-450 reductase from house fly microsomes were obtained in rabbits. Evolution of antibody titre was determined by the increasing inhibition of house fly microsomal NADPH-cytochrome c reductase activity by serum samples. An IgG fraction was obtained from the serum by ammonium sulphate precipitation. These antibodies inhibited microsomal NADPH-cytochrome c reductase from insecticide-resistant and susceptible house fly strains as well as from phenobarbital-treated insects. The antibodies were specific for NADPH-cytochrome P-450 reductase because they did not inhibit the NADH-dependent reduction of cytochrome c, dichlorophenolindophenol or ferricyanide. The antibodies distinguished the native NADPH-cytochrome P-450 reductase from the proteolytically processed NADPH-cytochrome c reductase as shown by differential inhibition of solubilized, purified enzymes. The antibodies inhibited microsomal cytochrome P-450 monooxygenase activities such as epoxidation and O- dealkylation , thus confirming the role of NADPH-cytochrome P-450 reductase in these reactions. Cross-reactivity towards the microsomal enzyme of 15 animal species was shown by inhibition of NADPH-cytochrome c reductase activity. Cross-reactivity was high for the enzyme of other Diptera such as a Drosophila melanogaster and Culex pipiens, but was very low for NADPH-cytochrome c reductase from vertebrate sources.