Amino-terminal sequence and structure of monoclonal antibody immunopurified cytochromes P-450.
Six hepatic cytochromes P-450 were isolated from 3-methylcholanthrene-treated animals by immunopurification with monoclonal antibodies. The purified cytochromes P-450 include 57- and 56-kDa polypeptides from Sprague-Dawley rats, 57- and 56-kDa polypeptides from C57BL/6 mice, a 56-kDa polypeptide from DBA/2 mice, and a 53-kDa polypeptide from guinea pigs. These isozymes were structurally compared by peptide mapping using both sodium dodecyl sulfate--polyacrylamide gel electrophoresis and high-pressure liquid chromatography and by amino acid and NH2-terminal sequence analyses. The 57-kDa polypeptides from rats and mice have similar but nonidentical peptide maps and amino acid compositions and are about 80% homologous in their NH2-terminal amino acid sequence. The 56-kDa polypeptides from rats and both mice strains have very similar peptide maps and amino acid compositions and identical NH2-terminal sequences. The NH2-terminal sequence of the mice 56-kDa polypeptides corresponds to that reported for the mouse P1-450 isozyme except that we identified two additional residues, proline and serine, at the NH2 terminus in the 57-kDa polypeptide from C57BL/6 mice that were not deduced from the cDNA sequence of the mouse P1-450 isozyme. The guinea pig 53-kDa polypeptide has a distinct peptide map relative to the other polypeptides studied and an NH2-terminal sequence with only partial homology to the 56- and 57-kDa polypeptides from rats and mice. This report shows the varying degree of structural relatedness among the isozymes examined and demonstrates the suitability and advantage of immunopurified cytochromes P-450 for sequencing and structural studies.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006168:Guinea Pigs; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System