AbstractsBiology & Animal Science

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

by Robert D. Schonbrod




Institution: Oregon State University
Department: Entomology
Degree: PhD
Year: 1971
Keywords: Chemical inhibitors
Record ID: 1489711
Full text PDF: http://hdl.handle.net/1957/45425


Abstract

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…