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The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes
Abstract
Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
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Cited by
Identification of Human Liver Cytochrome P450 Isoforms Mediating Omeprazole Metabolism
ANDERSSON, T., MINERS, J. O., VERONESE, M. E., TASSANEEYAKUL, W., TASSANEEYAKUL, W., MEYER, U. A., & BIRKETT, D. J. (1993) · British Journal of Clinical Pharmacology · doi:10.1111/j.1365-2125.1993.tb00410.x · Cited by 230
Andersson, T., Miners, J., Veronese, M., & Birkett, D. (1994) · British Journal of Clinical Pharmacology · doi:10.1111/j.1365-2125.1994.tb04336.x · Cited by 225
Multiple forms of human P450 expressed in Saccharomyces cerevisiae
Imaoka, S., Yamada, T., Hiroi, T., Hayashi, K., Sakaki, T., Yabusaki, Y., & Funae, Y. (1996) · Biochemical Pharmacology · doi:10.1016/0006-2952(96)00052-4 · Cited by 207
(2001) · PubMed · Cited by 163
Xuan, J., Chen, S., Ning, B., Tolleson, W. H., & Guo, L. (2016) · Chemico-Biological Interactions · doi:10.1016/j.cbi.2015.10.009 · Cited by 103
Metadata
- Authors
- Yamano, S., Tatsuno, J., & Gonzalez, F. J.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1990-02-06
- Volume
- 29
- Issue
- 5
- Pages
- 1322-1329
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2007972960
- Cited by
- 383
- References
- 0
- FWCI
- 18.23
- Type
- journal article
- Language
- en
- Open access
- closed
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- Yamano, S., Tatsuno, J., & Gonzalez, F. J. (1990). The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry. https://doi.org/10.1021/bi00457a031
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