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.
Roby
K F
KF
Department of Physiology, Ralph L. Smith Mental Retardation Research Center, University of Kansas Medical Center, Kansas City 66103.
Larsen
D
D
Deb
S
S
Soares
M J
MJ
D000311:Adrenal Glands / Q000166:cytology / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000032:analysis* / Q000276:immunology; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis*; D007150:Immunohistochemistry; D008647:Mesocricetus; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000276:immunology; D011817:Rabbits; D051381:Rats; D013552:Swine
D000939:Epitopes; D010455:Peptides; D002786:Cholesterol Side-Chain Cleavage Enzyme