Identification of a common cytochrome P450 epitope near the conserved heme-binding petide with antibodies raised against recombinant cytochrome P450 family 2 proteins.
The cytochrome P450 (P450) proteins constitute a superfamily of enzymes involved in various oxidations and related activities. Polyclonal antibodies raised against bacterial recombinant human P450s varied in specificity, depending upon the individual rabbits used. Several of the antisera raised against P450s 2C10 and 2E1 recognized a number of P450 family 1, 2, and 3 proteins, and two of the less selective antibodies were used to identify cross-reactive epitopes. P450 2C10 peptides reacting with anti-P450 2E1 and P450 2E1 peptides reacting with anti-P450 2C10 were isolated after electrophoresis/immunoblotting and analyzed by Edman degradation. Several of these were in a region near the highly conserved Cys that is a putative axial ligand to the heme. Peptides corresponding to the most conserved regions in this area were synthesized. Anti-P450 2C10 sera did not recognize 14-mer peptides corresponding to the heme-binding region (2C10 410-423 or 2E1 409-422) or the 14-mer peptides immediately C-terminal to these (2C10 425-438 or 2E1 424-437), but anti-P450 2E1 sera showed weak reaction with the latter two synthetic peptides. A longer peptide (29-mer) of P450 2E1 containing parts of both regions (412-440) reacted with both anti-P450 2C10 and anti-P450 2E1 antisera. Antibodies raised against a conjugate of the 29-mer peptide (with hemocyanin) recognized this antigen, the more C-terminal 14-mer peptides (2C10 425-438 and 2E1 424-437), P450s 2C10 and 2E1, and P450s 1A1, 11A1, and 17A. The 29-mer peptide showed considerable alpha-helix structure as judged by CD spectroscopy, in contrast to any of the 14-mers.(ABSTRACT TRUNCATED AT 250 WORDS)
Soucek
P
P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Martin
M V
MV
Ueng
Y F
YF
Guengerich
F P
FP
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D016384:Consensus Sequence; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D004851:Epoxide Hydrolases / Q000737:chemistry; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits / Q000276:immunology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D000939:Epitopes; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D004851:Epoxide Hydrolases