Characterization of NADPH-cytochrome P-450 reductase from house flies (Musca domestica,L.) susceptible and resistant to insecticides,and the blow fly (Phormia regina,meigen)

NADPH-cytochrome $\text{c}$ reductase was purified by affinity chromatographic techniques from microsomes prepared from the abdomens of insecticide-resistant (R) and -susceptible (S) house flies Musca domestica and of the black blow fly Phormia regina. Data are presented which describe (1) the ability of the purified enzymes to support an in vitro reconstitution of mono-oxygenase activity, (2) the changes in activity of these preparations observed in buffers of varying ionic strength, (3) comparative kinetic behaviour between microsomal and purified forms of the enzymes, (4) the immunochemical characteristics of these preparations, and (5) their amino acid composition. The reductases from the three sources were found to be very similar in all of these tests. Substrate binding constants were 5 μM for NADPH, 12 μM for cytochrome $\text{c}$, and the catalytic mechanism was interpreted as ordered Bi Bi. The inhibitory constant of the reductase from the resistant fly for 2′-AMP, an analogue of NADP⁺, was 187 μM; whereas no assciation of the inhibitor was observed below concentrations of 400 μM for the enzyme of either the susceptible house fly or the blow fly. However, the data are insufficient to suggest that the reductase is a significant factor in insecticide resistance. Compared to the same enzymes from rat and rabbit liver, the insect reductases show a different ionic strength optimum (0.14), have distinct antigenic determinants, and have different levels of acidic and basic amino acids in the membrane-binding peptide.