Interaction of microsomal cytochrome P450 isozymes isolated from wild-type and DMI-resistant isolates of Penicillium italicum with DMI fungicides

Microsomal cytochrome P450 isozymes were isolated from isolates of Penicillium italicum with various degrees of resistance to fungicides which inhibit sterol 14 alpha-demethylation (DMIs). The yield of P450 isozymes isolated from the low-resistant isolate E(300-3) was similar as that from wild-type isolate W5 (365 pmol g-1 dry wt mycelium) whereas the yield from medium- and high-resistant isolates H17, I33, and J4 was significantly lower (285, 301, and 223 pmol g-1 dry wt, respectively). The specific content of P450 isozymes per milligram microsomal protein from isolates H17, I33, and J4 was also relatively low (50, 52, and 34 pmol mg-1 protein, respectively) compared with that of isolate W5, (95 pmol mg-1 protein). This may be an intrinsic characteristic of these isolates, but can also be a consequence of slight modifications in the standard isolation procedure as described for isolate W5. Maximum absorbance of the reduced carbon monoxide (CO) difference spectrum of P450 isozymes of isolate E(300-3) was at 449 nm, which is identical to that of isolate W5. Isolates H17 and I33 had a maximum absorbance at 450 nm and isolate J4 at 452 nm, respectively. The content of P420 isozymes in isolates H17, I33, and J4 was higher than in microsomes of isolates W5 and E(300-3). The oxidized form of the P450 isozymes of all DMI-resistant isolates interacted at equimolar concentration (10(-7) M). With imazalil (type II binding). The affinity of the heme iron in P450 isozymes in isolates H17, I33, and J4 to imazalil may be slightly lower than in isolates W5 and E(300-3) but can not account for the mechanism of resistance. In CO-displacement tests imazalil, itraconazole, and ketoconazole were more readily displaced from P450 isozymes of isolates H17, I33, and J4 than from isozymes of isolates W5 and E(300-3). At first glance, these results suggest that one of the cytochrome P450 isozymes from medium- and high-resistant isolates, possibly cytochrome P450(14DM), may have an altered apoprotein, resulting in decreased affinity to DMIs. However, the quality of microsomal P450 preparations of the isolates (e.g., protein content, P420 content, stability of P450 isozymes in presence of DMIs, cytochrome P450 composition, and contamination with cytochrome oxidase) between wild-type and medium- and high-resistant isolates differs significantly. These differences will not only affect the spectrophotometric properties of P450 isozymes but also the CO displacement rate of DMIs from the isozymes and therefore hamper a proper comparison of the results. It is concluded that microsomal cytochrome P450 isozymes cannot be used to compare the spectrophotometric properties and the affinity of cytochrome P450(14DM) from different isolates of P. italicum to DMIs. This conclusion may be of general significance for similar studies with P450 isozymes of other organisms.