Substrate influence on interaction between cytochrome P450 and cytochrome b5 in microsomes.

We have been able to demonstrate that cytochrome b4 interacts closely with cytochrome P450 in the microsomal membrane, and that substrates can serve to order the interaction: Using the water-soluble carbodiimide EDC we could crosslink cytochrome b5 and CYP2B4 in microsomes from phenobarbital-treated rabbits and cytochrome b5 and CYP1A2 in microsomes from beta-naphthoflavone-treated animals. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, decreasing the formation of higher molecular weight oligomeric complexes with cytochrome b5. In contrast, no ordering of the interactions were obtained on addition of 7-ethoxycoumarin, a substrate of CYP1A2, or of benzphetamine to microsomes of beta-naphthoflavone-treated animals in the presence of EDC. Of interest, although evidence could be shown for complementary charge-pairing between cytochrome b5 and a number of other microsomal proteins in the membranes, and while the extent of CYP1A2 and CYP2B4 interaction with cytochrome b5 each exceeded 30% in the presence of substrate, no significant complexation of the P450s was obtained with any other microsomal proteins.