Dissecting the function of cytochrome P450.
1. The role that P450 plays in the metabolism of drugs and other chemicals is often pivotal in determining the duration and magnitude of their biological effects, so that it is important to identify the specific forms of the enzyme involved. 2. The similarity between different forms of P450 is such that polyclonal antibodies raised by conventional means, even against a homogeneous preparation of the enzyme will often react with several different P450s. 3. Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to their use. 4. The use of short sequences of amino acids (4-7 residues) predicted to occur in surface loop regions of the protein as immunogens, affords a means of targeting antibodies to specific forms of P450. 5. The terminal amino acid of a peptide, here the C-terminal residue, is very immunodominant in determining the specificity of the resultant antibody. Hence, antibodies against the C-terminal residues of P450 have a high affinity and, in most cases, will be specific. 6. Application of anti-peptide antibodies to the major forms of P450 in human liver revealed the anticipated interindividual variation. However, evidence was found that the polymorphism in CYP3A5 expression is quantitative rather than qualitative. 7. A putative pro-inhibitory region on the surface of mammalian P450, possibly involved in intramolecular electron transport, has been identified, using a combination of directed anti-peptide antibodies and sequence alignment based on secondary structure. 8. Antibodies directed against defined regions of P450 enzymes are proving invaluable in exploring the regulation, specificity and function of these key enzymes.
Boobis
A R
AR
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Edwards
R J
RJ
Adams
D A
DA
Davies
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System