Probing the putative cytochrome P450- and cytochrome c-binding sites on NADPH-cytochrome P450 reductase by anti-peptide antibodies.
Two regions (amino acid residues 110-130 and 204-218) of NADPH-cytochrome P450 reductase (reductase) have been shown to be the putative binding sites for the interaction with cytochrome P450 or cytochrome c. To obtain further insight into the molecular mechanism of protein-protein interaction between these proteins, three anti-peptide antibodies (1A, 2A, and 3A) were generated against the peptides corresponding to these 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 (titre > 1 x 10(-6) g/L), and they also bind to rat reductase on ELISA under both denatured and native conditions, suggesting that these regions are on the surface of the protein. 1A and 3A also bind to rabbit and human 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 ethoxycoumarin hydroxylation activity and P4502B1-dependent pentoxyresorufin dealkylation activity, but the inhibition by 1A and 3A was not additive. Antibodies 1A and 3A also have inhibitory effects on the activity of P4501A1-dependent ethoxycoumarin hydroxylation reconstituted with reductase from rabbit and human. However, none of the three anti-peptide antibodies inhibits cytochrome c reduction by rat reductase. These data suggest that reductases from rat, rabbit, and human share similar structure in at least two regions which appear to be on the surface of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
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D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D051381:Rats
D003574:Cytochrome c Group; D010455:Peptides; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase