PROBING THE PUTATIVE CYTOCHROME-P450- AND CYTOCHROME-C BINDING SITES ON NADPH-CYTOCHROME-P450 REDUCTASE BY ANTIPEPTIDE ANTIBODIES

Two regions (amino acid residues 110-130 and 204-218) of NADPH-cytochr ome P450 reductase (reductase) have been shown to be the putative bind ing sites for the interaction with cytochrome P450 or cytochrome c. To obtain further insight into the molecular mechanism of protein-protei n interaction between these proteins, three anti-peptide antibodies (1 A, 2A, and 3A) were generated against the peptides corresponding to th ese two regions on rat reductase to study the interaction between the reductase and cytochrome P450 or cytochrome c. All three anti-peptide antibodies have high affinity for their peptide antigens on ELISA (tit re > 1 X 10(6) g/L), and they also bind to rat reductase on ELISA unde r both denatured and native conditions, suggesting that these regions are on the surface of the protein. 1A and 3A also bind to rabbit and h uman reductase, though 1A binds to human reductase with lower affinity . Antibody 2A does not bind to rabbit or human reductase. Western blot analysis using these anti-peptide antibodies showed similar results. Antibodies 1A and 3A inhibit both cytochrome P4501A1-dependent ethoxyc oumarin hydroxylation activity and P4502B1-dependent pentoxyresorufin dealkylation activity, but the inhibition by 1A and 3A was nor additiv e. Antibodies 1A and 3A also have inhibitory effects on the activity o f P4501A1-dependent ethoxycoumarin hydroxylation reconstituted with re ductase from rabbit and human. However, none of the three anti-peptide antibodies inhibits cytochrome c reduction by rat reductase. These da ta suggest that reductases from rat, rabbit, and human share similar s tructure in at least two regions which appear to be on the surface of the protein. These two regions (110-119 and 204-218) of rat reductase both seem to be involved in the interaction with cytochrome P450, whil e the association of reductase with cytochrome c may depend on differe nt mechanisms.