An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity.
An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, U.K.
R J
RJ
Edwards
A M
AM
Singleton
B P
BP
Murray
D
D
Sesardic
K J
KJ
Rich
D S
DS
Davies
A R
AR
Boobis
D015073:2-Acetylaminofluorene / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology* / Q000378:metabolism; D004790:Enzyme Induction; D000939:Epitopes; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D051381:Rats
D000939:Epitopes; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System; D015073:2-Acetylaminofluorene; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2