Two monoclonal antibodies recognizing different epitopes on rat cytochrome IIB1 react with human IIE1.
To identify human cytochromes P450 (P450) in the CYP2B subfamily, 14 human liver microsomal samples were screened by immunoblots developed with monoclonal antibodies that recognized seven distinct epitopes on rat IIB1. Two of these antibodies recognized a protein in all of the samples. This protein was termed P450BE. Using video-imaging densitometry, the levels of P450BE were determined and compared with levels of other P450s. An excellent correlation was seen (r = 0.87) between P450BE and human IIE1. However, rat IIE1 did not react in immunoblot and enzyme-linked immunosorbant assays with the two anti-rat IIB1 monoclonal antibodies. As previously observed, the levels of IIE1 in the samples correlated well (r = 0.88) with the ability of these human liver microsomes to N-demethylate N-nitrosodimethylamine. The levels of P450BE also correlated well (r = 0.91) with the ability of the microsomes to N-demethylate N-nitrosodimethylamine. In addition, excellent correlations were obtained when the levels of P450BE and IIE1 were compared with the ability of the microsomes to O-deethylate ethoxycoumarin (r = 0.87 and r = 0.85, respectively). To identify the protein recognized by the anti-rat IIB1 antibodies, P450BE was purified from microsomes prepared from human liver D. Amino-terminal amino acid sequence analyses of P450BE revealed that the 18-amino acid sequence obtained matched the corresponding sequence of human IIE1. In addition, purified human IIE1 and P450BE migrated with the same apparent molecular weight in polyacrylamide gels. Furthermore, proteolytic maps of P450BE and IIE1, generated with two proteases, were found to be identical. Sequence alignments and antigenicity calculations identified three regions of rat IIB1 as likely candidates for the epitopes shared in common with human IIE1. In conclusion, this study indicates that caution must be taken when interpreting the results of immunochemical assays when species lines are crossed.
Department of Drug Metabolism and Disposition, Lilly Corporate Center, Indianapolis, Indiana 46285.
S A
SA
Wrighton
M
M
Vandenbranden
G W
GW
Becker
S D
SD
Black
P E
PE
Thomas
D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D019362:Cytochrome P-450 CYP2B1; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D003720:Densitometry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008875:Middle Aged; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000276:immunology* / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000276:immunology* / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019392:Cytochrome P-450 CYP2E1; D019362:Cytochrome P-450 CYP2B1; D010089:Oxidoreductases, N-Demethylating