Abstract
Cytochrome P450 (CYP) 2E1 is a toxicologically important enzyme that inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Although cDNAs for the human, rodent, and rabbit forms of CYP2E1 have been isolated and studied extensively, there is an absence of information about canine CYP2E1, despite the fact that the dog is routinely used in drug safety studies. In this study, we isolated and sequenced a full-length CYP2E1 cDNA from a beagle liver cDNA library. The deduced canine CYP2E1 amino acid sequence exhibited 75 to 76% identity with rat, mouse, and rabbit CYP2E1 sequences, and 77% identity with human CYP2E1. Two populations of clones, differing at a single nucleotide, were isolated from the unamplified library. The T1453C base change results in a Tyr485His amino acid substitution, which is well beyond the heme binding region but is possibly part of a β-sheet structure. An allele-specific polymerase chain reaction-based restriction enzyme test was developed for genotyping individual dogs from genomic DNA samples. One hundred mixed breed dogs were genotyped, and the frequencies of the Tyr485 and His485 alleles were found to be 0.85 and 0.15, respectively. The canine Tyr485 and His485 alleles and human CYP2E1 were expressed inEscherichia coli cells, and catalytic activities of the proteins were assessed using the substrate chlorzoxazone. Although the two canine enzymes had similar catalytic activity; significant kinetic differences were seen between canine and human CYP2E1s.
Footnotes
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Send reprint requests to: Susan M. Lankford, Department of Anatomy, Physiological Sciences and Radiology, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough St., Raleigh, NC 27606. E-mail: susan-lankford{at}ncsu.edu
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↵1 The nucleic acid sequences in this paper have been submitted to GenBank under accession numbers AFO29978 and AFO29979.
- Abbreviations used are::
- CYP
- cytochrome P450
- bp
- base pair(s)
- 4-MP
- 4-methylpyrazole
- PCR
- polymerase chain reaction
- Received February 3, 2000.
- Accepted May 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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