Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca |
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Authors: | Anne E. Desjardins Hans D. VanEtten |
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Affiliation: | (1) Department of Plant Pathology, Cornell University, 14853-0331 Ithaca, NY, USA;(2) Present address: Northern Regional Research Center, USDA, 1815 N. University Street, 61604 Peoria, IL, USA |
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Abstract: | The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts. |
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Keywords: | Cytochrome P-450 Nectria haematococca Phytoalexin Detoxification |
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