title: Isolation of a bovine full length cytochrome P450 (CYP3A) cDNA sequence and its functional expression in V79 cells.
abstract: From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6β-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6β-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2β and 12β position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.
authors.affiliation: Department of Veterinary Pharmacology, Utrecht University, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Natsuhori
authors.forename: M
authors.initials: M
authors.lastname: van Raak
authors.forename: M
authors.initials: M
authors.lastname: Ligtenberg
authors.forename: L
authors.initials: L
authors.lastname: Kleij
authors.forename: D
authors.initials: D
authors.lastname: Ten Berge
authors.forename: W M
authors.initials: WM
authors.lastname: Zweers-Zeilmaker
authors.forename: E M
authors.initials: EM
authors.lastname: de Groene
authors.forename: A S
authors.initials: AS
authors.lastname: van Miert
authors.forename: R F
authors.initials: RF
authors.lastname: Witkamp
authors.forename: G J
authors.initials: GJ
authors.lastname: Horbach