DISSECTING THE FUNCTION OF CYTOCHROME-P450

1 The role that P450 plays in the metabolism of drugs and other chemic als is often pivotal in determining the duration and magnitude of thei r biological effects, so that it is important to identify the specific forms of the enzyme involved. 2 The similarity between different form s of P450 is such that polyclonal antibodies raised by conventional me ans, even against a homogeneous preparation of the enzyme will often r eact with several different P450s. 3 Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to t heir use. 4 The use of short sequences of amino acids (4-7 residues) p redicted 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 a ntibody. Hence, antibodies against the C-terminal residues of P450 hav e 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 r evealed the anticipated interindividual variation. However, evidence w as found that the polymorphism in CYP3A5 expression is quantitative ra ther than qualitative. 7 A putative pro-inhibitory region on the surfa ce of mammalian P450, possibly involved in intramolecular electron tra nsport, has been identified, using a combination of directed anti-pept ide antibodies and sequence alignment based on secondary structure. 8 Antibodies directed against defined regions of P450 enzymes are provin g invaluable in exploring the regulation, specificity and function of these key enzymes.