Graduate Thesis Or Dissertation
 

Biochemical properties of an endogenous inhibitor of house fly (Musca domestica L.) microsomal oxidations

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  • Endogenous inhibitors of insect microsomal oxidases are a serious problem in in-vitro studies of insecticide metabolism. Their number, chemical nature, modes of action, and their effectiveness as inhibitors are unknown. In the case of house flies, there may be as many as four inhibitors. The most important of these is known to be heat stable and to be found mainly in the head of the fly. This inhibitor was investigated to determine its identity and mode of action. The inhibitor as assayed by its effect on microsomal aldrin epoxidation. Fly heads were homogenized with distilled water, heated, filtered and used as inhibitor preparations. Gas-liquid chromatography was used to analyze for dieldrin, the product of the enzyme reaction. The activities of microsomal NADPH:cytochrome C oxidoreductase and NADPH:neotetrazolium oxidoreductase were measured spectrophotnmetrically using cytochrome C, 2,6-dichlorophenolindophenol (DCPIP), and neotetrazolium as substrates. The house flies used in these experiments differed genetically in respect to their level and type of insecticide resistance, their microsomal oxidase level, and their eye color. The head inhibitor did not affect the stability of the epoxidase enzyme since the percent inhibition was constant at various times of incubation. Double reciprocal plots of reaction velocity versus substrate concentration showed that the inhibitor is not a competitor with aldrin for the same active site on the enzyme. According to these methods, the inhibitor does not compete with NADPH for the site of reaction, but it does reduce the activity of the microsomal electron transport system. The amount of head inhibitor was not dependent on the age, sex, nature of insecticide resistance, or microsomal oxidase activity of the fly. However, the inhibitor was absent in house fly strains with the white and ocra eye color mutations and was present at a reduced level in the flies with carmine colored eyes. These results showed that the pigment required for wild type eye coloration is directly involved with the inhibitor. This pigment, xanthommatin, a product of tryptophane metabolism, is absent in strains with genetic blocks at the third and fifth chromosomes. Xanthommatin was obtained by synthesis and by isolation from fly heads. When included at 5 x 10⁻⁶ M and 5 x 10⁻⁷ M in microsome incubations, xanthommatin inhibited the activity of the epoxidase enzyme 72.5% and 17.5%, respectively. The precursor of xanthommatin, 3-hydroxykynurenine, was not inhibitory. Xanthommatin increased the rate of oxidation of NADPH by the microsomes, as did cytochrome C, DCPIP, and neotetrazolium. Because dihydroxanthommatin is rapidly air-oxidized, the reduction of xanthommatin by NADPH and microsomes could be detected only under anaerobic conditions, it was concluded that xanthommatin serves as an electron acceptor for the microsomal electron transport system, limiting the supply of electrons to the epoxidase system. The inhibitory effect of xanthommatin is enhanced by the auto-oxidation of dihydroxanthommatin which provides additional electron acceptor. A double-reciprocal plot of enzyme reaction velocity versus substrate concentration showed that xanthommatin inhibits neotetrazolium reduction non-competitively, The data indicate that the inhibitor limits the flow of reducing potential to the non-heme iron protein, Similar experiments with NADPH:cytochrome C oxidoreductase showed a "mixed" inhibition, Xanthommatin appeared to increase the rate of cytochrome C reduction, but this increase was found to be non-enzymic. It is suggested that the site of action of xanthommatin is NADPH: cytochrome C oxidoreductase. When tested in epoxidase incubations which contained the inhibitor, bovine serum albumin (BSA) did not affect the percent inhibition, The effect of xanthommatin on the electron transport components was not altered by the presence of BSA. However, BSA decreased the reduction of DCPIP approximately 30% when used at a level of 1 to 2 mg/fly equivalent of microsomes. This same concentration of BSA was found to be optimum for maximum enhancement of microsomal aldrin epoxidation. The minimum increase of epoxidase activity due to BSA occurred under optimum conditions of incubation. It was concluded that BSA does not counteract the inhibitory effect of xanthommatin, and it is suggested that the enhancing effect of BSA on the microsomal oxidases is due to an influence on the configuration of the microsome.
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