Characteristics of NADPH-cytochrome P-450 reductase in the house fly (Musca domestica, L.) and the blow fly (Phormia regina, Meigen) Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/5138jj84r

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  • NADPH-cytochrome P-450 reductase, an important enzyme in the cytochrome P-450-dependent microsomal monooxygenase system, was purified from insecticide-resistant (R) and -susceptible (S) house flies (Musca domestica, L.) and from the black blow fly (Phormia regina, Weigen). Proteolytic degradation to a P-450 - inactive form of the reductase was reduced during the procedure by the use of specific protease inhibitors (leupeptin and chymostatin) as well as by careful timing of other key steps. Following the several steps in the procedure, the homogeneous enzymes (M[subscript r] = 74,000 for either strain of M. domestica, and 75,000 for P. regina) was purified ca. 360-fold, achieving specific activities ranging from 32 to 40 μmol cytochrome c reduced/min/mg protein. Each of these purified enzymes was competent in supporting either epoxidation or 0-demethylation reactions involving soluble house fly cytochrome P-450 and synthetic phospholipid. Comparisons of kinetic behavior, absolute spectra, response to ionic strength, amino acid composition, or immunochemical cross-reactivity indicated that the three reductases were nearly identical. However, the purified R-fly enzyme was more sensitive to the NADPH analogue, 2'-AMP, (Ki = 187 μM) than the other two reductases. These insect enzymes were distinctively different from rat or rabbit liver P-450 reductases in their response to ionic strength, sensitivity to 2'-AMP, amino acid composition, and antigenic determinants. Microsomal reductase activity was lowest in pharate adult flies (ca. 50 μmol product/min/mg protein) and increased to a maximum (ca. 120 μm.' product/min/mg protein) 24 to 48 hr after emergence. The activity then assumed a rather constant value (ca. 80 μmol product/min/mg protein) through 5 days of age. These values were similar in the three insects. Treatment of the three flies with phenobarbital, a microsomal monooxygenase inducer, caused the reductase activity to increase about 2.5-fold in the two house fly strains and 5-fold in the blow fly strain. P-450 also increased several fold, as did monooxygenase activities.
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