Amino-terminal sequence and structure of monoclonal antibody-immunopurified cytochromes P 450

Abstract

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.

Human Liver Cytochrome P-450 Related to a Rat Acetone-Inducible, Nitrosamine-Metabolizing Cytochrome P-450: Identification and Isolation

“…Monoclonal antibodies have proven use ful in several key phases of P-450 research [3,4], The MAb-based immunopurification in particular has provided a means to rea dily obtain liver P-450s in a one-step col umn procedure [5][6][7], These P-450s are quite suitable for primary structural analy sis and have yielded new information on comparative structure of liver P-450s from different strains and species [7], as well as a rat lung P-450 [8]. Although these MAb immunopurifications were performed on ani mal tissues, the MAbs to rat P-450s also interact with P-450 in human tissues as de termined by activity inhibition data [34][35][36] and radioimmunoassay [37].…”

“…These immunopurified P-450s were relatively resistant to proteolytic diges tion when compared to other forms of P-450 we have previously digested [7] and required prolonged incubation with a high ratio (1:10) of enzyme to protein. This indicates that the target cleavage sequences are partly inacces sible, possibly owing to conformational limi tations or aggregation of the polypeptides.…”

“…MAbs to different P-450s have been successfully uti lized for radioimmunoassay, enzyme inhibi tion assay and for immunopurification of P-450s from a variety of sources [3,4], Progress toward purification of a large number of P-450s has been hindered by the difficulties encountered in conventional pu rification procedures which are lengthy, may have low yields and often are not applicable to the purification of cytochromes P-450 present in low concentrations. A significant advance in this field has been the application of MAbs for immunopurification of P-450s from animal tissues [5][6][7][8]. Although human liver P-450s have been purified by the usual conventional procedures [9][10][11][12][13], these have not heretofore been subjected to the MAb immunopurification methods.…”