Testosterone hydroxylation in bovine liver: enzyme kinetic and inhibition study.
In order to sort out the involvement of cytochrome P450 (CYP) 3A and possibly CYP2B in testosterone hydroxylation in cattle, enzyme kinetic and inhibition studies were performed. Most relevant kinetic constants (Km and Vmax) for 6beta-, 16beta- and 2beta-testosterone hydroxylase (OHT) activities were determined and accounted for 93.4 +/- 13.8, 36.4 +/- 6.1 and 110.8 +/- 15.2 muM, respectively, for Km and 0.558 +/- 0.03, 0.280 +/- 0.013, and 0.338 +/- 0.017 nmol min-1 mg-1 protein, respectively, for Vmax. Eadie-Hofstee plot analysis pointed out how these enzymatic activities in cattle follow a monophasic kinetic pattern. Preliminary inhibition studies conducted with the CYP3A inhibitor ketoconazole and the CYP2B inhibitors orphenadrine and 9-ethynylphenanthrene seemed to suggest the major involvement of CYP3A in testosterone hydroxylation in cattle. Immuno-inhibition studies with an anti-peptide antibody against bovine CYP3A4 confirmed the predominant role of CYP3A in testosterone hydroxylation in bovine liver, proving the usefulness of anti-peptide antibodies in defining the contribution of specific P450 isoforms in drug metabolism in veterinary species.
Pegolo
S
S
Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Viale dell'Università 16, Legnaro, Padua, Italy.
Giantin
M
M
Dacasto
M
M
Montesissa
C
C
Capolongo
F
F
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A / Q000276:immunology / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006900:Hydroxylation; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000276:immunology; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism*
D000906:Antibodies; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D013739:Testosterone; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C025619:steroid 16-beta-hydroxylase; C022657:steroid hormone 6-beta-hydroxylase; D007654:Ketoconazole