Interaction of microsomal cytochrome P450 isozymes isolated from Penicillium italicum with DMI fungicides
1992
Guan, J. | Braks, H.M.J. | Kerkenaar, A. | Waard, M.A. de
A procedure has been developed for the isolation of microsomal cytochrome P450 isozymes from the filamentous fungus Penicillium italicum. The content of cytochrome P450 isozymes in the microsomal fraction was 95 +/- 18 pmol/mg protein. Maximum absorbance of the reduced carbon monoxide (CO) difference spectrum was at 449 nm. Absorbance at 420 nm was absent. The fungitoxic sterol 14alpha,-demethylation inhibitors (DMIs) imazalil, itraconazole, ketoconazole, penconazole, and propiconazole and the two less-toxic imazalil analogues R14821 and R42243 interacted with oxidized cytochrome P450 isozymes resulting in type II binding difference spectra. Equimolar concentration (10 - 7 M) of cytochrome P450 isozymes and DMIs induced about 80% of the maximum absorbance difference, indicating a close stoichiometric interaction. The concentration of DMIs which induced 50% of the maximum absorbance difference in the type II spectra (IC50) ranged from 3.7 to 5.0 X 10-8 M and did not correlate with fungitoxicity of the DMIs. Imazalil analogues did not interact stoichiometrically with cytochrome P450 isozymes and gave relatively higher lC50 Values. The binding of the DMIs and imazalil analogues to cytochrome P450 isozymes was studied by CO displacement tests. The ease of displacement of the test compounds decreased in the order of R42243, R14821, penconazole, propiconazole, and imazalil. This correlated with the fungitoxicity of these compounds. However, DMIs with a relatively large N-1 substituent like itraconazole and ketoconazole did not show such a correlation, indicating that factors, in addition to binding affinity to the heme iron and apoprotein of cytochrome P450 isozymes, also influence toxicity.
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