Dissecting the function of cytochrome P450

Abstract

1The 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. 2The 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. 3Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to their use. 4The 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. 5The 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. 6Application 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. 7A 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. 8Antibodies directed against defined regions of P450 enzymes are proving invaluable in exploring the regulation, specificity and function of these key enzymes.

African variation at Cytochrome P450 genes

“…There is considerable overlap between CYP3A enzymes in substrate-specificity [ 32 ]. Many previous studies have argued that CYP3A4 has the most significant role of all CYP3A enzymes in therapeutic drug metabolism [ 33 ]. However, CYP3A5 is one of the most pharmacologically active drug metabolizing enzymes in Africa [ 29 ].…”

Pharmacokinetics of Midazolam and 1′-Hydroxymidazolam in Chinese with DifferentCYP3A5Genotypes

“…Another study showed that only 3% of human liver had detectable CYP3A5 by immunoblotting (Boobis et al, 1996). These findings suggested that CYP3A5 polymorphism might play an important role in functional regulation.…”

Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry

“…7 The quantification methods that are most widely used today are immunological techniques such as ELISA and Western blotting 8 which rely on the availability of specific antibodies. Although tremendous efforts have been made to develop isoform-specific monoclonal 9 or anti-peptide antibodies, 10 the simultaneous and comparative quantification of CYP enzymes is still a very difficult task because of their high similarities both in sequence and in molecular weight. For example, recent data on the relative expression levels of CYP3A4 and CYP3A5 have remained controversial.…”