|Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes.|
Edwards, R J, et al.
Biochem. Pharmacol., 49: 39-47 (1995)
An antibody was raised against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn) corresponding to residues 290-296 of the cytochrome P450 enzyme, CYP1A2, of both rat and mouse. A cysteine residue attached to the N-terminus of the peptide during synthesis allowed coupling in a specific orientation via the thiol group to the carrier protein, keyhole limpet haemocyanin. Antiserum raised in rabbits bound specifically to CYP1A2 in the rat and mouse. To determine those amino acid residues involved in binding of the antibody, related peptides of various lengths were synthesised and the binding of the antibody was determined by an enzyme-linked immunosorbent assay. These studies show that the minimum epitope is the C-terminal tripeptide sequence, Lys-Asp-Asn. Other than in rat and mouse CYP1A2, this tripeptide is found as an internal sequence in a large number of proteins including bovine fibronectin, chicken gizzard myosin heavy chain, and the P450 enzymes, rabbit CYP3A6 and human CYP3A4, but the antibody did not bind to any of these proteins. However, the antibody did bind to yeast glucose-6-phosphate dehydrogenase in which the tripeptide sequence is the C-terminus. Antibodies raised against a truncated peptide (Tyr-Lys-Asp-Asn), representing the C-terminal half of the peptide, also bound to glucose-6-phosphate dehydrogenase, but failed to bind to CYP1A2; thus although the C-terminal region of the peptide 290-296 is strongly immunogenic, it appears that it is not this population of antibodies that binds to CYP1A2. As antibodies were found to bind strongly to the C-terminus of glucose-6-phosphate dehydrogenase, the C-termini of proteins as targets for anti-peptide antibodies were investigated further by immunising rabbits with four 5-residue peptides which represent the C-termini of the P450 enzymes, CYP1A1, CYP1A2, CYP2E1 and CYP2A6. The peptides were coupled to keyhole limpet haemocyanin through their N-termini via cysteine residues added to the sequences. All four antisera bound specifically to their respective target proteins, as demonstrated by immunoblotting using hepatic microsomal fractions from rat, rabbit and human. It is suggested that this method of antibody production could be of general use for the reliable production of antisera against proteins where their sequence at the C-terminus is known, and such antibodies can be highly specific as they do not bind to internal sequences.
|The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature.|
Nelson, D R, et al.
DNA Cell Biol., 12: 1-51 (1993)
We provide here a list of 221 P450 genes and 12 putative pseudogenes that have been characterized as of December 14, 1992. These genes have been described in 31 eukaryotes (including 11 mammalian and 3 plant species) and 11 prokaryotes. Of 36 gene families so far described, 12 families exist in all mammals examined to date. These 12 families comprise 22 mammalian subfamilies, of which 17 and 15 have been mapped in the human and mouse genome, respectively. To date, each subfamily appears to represent a cluster of tightly linked genes. This revision supersedes the previous updates [Nebert et al., DNA 6, 1-11, 1987; Nebert et al., DNA 8, 1-13, 1989; Nebert et al., DNA Cell Biol. 10, 1-14 (1991)] in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene and cDNA, we recommend that the italicized root symbol "CYP" for human ("Cyp" for mouse), representing "cytochrome P450," be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. "P" ("p" in mouse) after the gene number denotes a pseudogene. If a gene is the sole member of a family, the subfamily letter and gene number need not be included. We suggest that the human nomenclature system be used for all species other than mouse. The mRNA and enzyme in all species (including mouse) should include all capital letters, without italics or hyphens. This nomenclature system is identical to that proposed in our 1991 update. Also included in this update is a listing of available data base accession numbers for P450 DNA and protein sequences. We also discuss the likelihood that this ancient gene superfamily has existed for more than 3.5 billion years, and that the rate of P450 gene evolution appears to be quite nonlinear. Finally, we describe P450 genes that have been detected by expressed sequence tags (ESTs), as well as the relationship between the P450 and the nitric oxide synthase gene superfamilies, as a likely example of convergent evolution.
|Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes.|
Thomas, P E, et al.
Fed. Proc., 46: 2563-6 (1987)
Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.