The cytochrome P-450 dependent monooxygenase activity in a 'wild type' susceptible strain of housefly was studied. Catalytic activities were identified that demonstrated that the housefly cytochrome P-450 was capable of similar catalytic functions as those described for higher animals. Although several specific activities were lower than in mammalian species, benzphetamine N-demethylation was comparable and there was higher constitutive activity toward lauric acid than is observed in rat hepatic microsomes. The inducing agent phenobarbital increased both total cytochrome P-450 content and the benzphetamine N-demethylase specific activity. The high constitutive activity for fatty acids was induced by the hypolipidaemic drug clofibrate, specifically inducing the w-hydroxylase activity. The substrate specificity toward lauric acid extended equally to myristic and palmitic acid. Housefly microsomal cytochrome P-450 also metabolised the unsaturated fatty acid, arachidonic acid, the w-hydroxy-lation again inducible by clofibrate pretreatment. The w-hydroxylation of these fatty acids appeared to be a well-coupled reaction, a property that also appeared to be exhibited by the rat hepatic w-hydroxylase. The housefly fatty acid hydroxylation showed certain similarities to that in the rat, both in the specificity for w-hydroxylation and in the result of induction by clofibrate. Structural comparison to cytochrome P-450IVA1, IIB1 and IA1 was made by Western blot analysis with polyclonal antibodies raised to these rat hepatic isoenzymes. Housefly cytochrome P-450 shared few, if any, common epitopes with these rat isoenzymes, nor did these antibodies inhibit housefly cytochrome P-450 dependent monooxygenase activity. Cytochrome P-450 from clofibrate-pretreated housefly microsomes was partially purified by affinity chromatography. The cytochrome P-450 had a specific content of 5. 7nmol.mg-1 and a monomeric molecular weight of 52,000 daltons and a reduced carbon monoxide difference spectrum absorbance maxima at 448nm. In a reconstituted system, this preparation exhibited activity toward lauric acid and to a lesser extent arachidonic acid in each case w-hydroxylated products predominated. This is the first example of a purification of an insect cytochrome P-450 multiple form with a defined product reaction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:234547 |
| Date | January 1989 |
| Creators | Clarke, Stephen Edward |
| Publisher | University of Surrey |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://epubs.surrey.ac.uk/847312/ |
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