Identification of a functionally conserved surface region of rat cytochromes P450IA.
A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp-Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de-ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450.
Edwards
R J
RJ
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, U.K.
Singleton
A M
AM
Murray
B P
BP
Murray
S
S
Boobis
A R
AR
Davies
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D019388:Cytochrome P-450 CYP1A2; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006433:Hemocyanins / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D065607:Cytochrome P-450 Enzyme Inhibitors; D000939:Epitopes; D007527:Isoenzymes; D006433:Hemocyanins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; C032808:keyhole-limpet hemocyanin