Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme.

In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P-450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes.