An inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome P-450 enzymes CYP1A1 and CYP1A2.
A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does not bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-Gln-Pro-Arg-Leu: residues 356-363 and 350-357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did not bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O-deethylase) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450, whilst binding of the monoclonal antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450. These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.
Edwards
R J
RJ
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Murray
B P
BP
Murray
S
S
Singleton
A M
AM
Davies
D S
DS
Boobis
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis; D001666:Binding Sites, Antibody; D006224:Cricetinae; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D011506:Proteins / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D065607:Cytochrome P-450 Enzyme Inhibitors; D000939:Epitopes; D010455:Peptides; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases