Testosterone hydroxylation in bovine liver: enzyme kinetic and inhibition study

Abstract

1. 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.

2. Most relevant kinetic constants (Km and Vmax) for 6β-, 16β- and 2β-testosterone hydroxylase (OHT) activities were determined and accounted for 93.4 ± 13.8, 36.4 ± 6.1 and 110.8 ± 15.2 μM, 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.

3. 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.

4. 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.

Keywords::

• Cattle
• testosterone
• K_m
• V_max
• inhibition studies
• anti-peptide antibody

Acknowledgements

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.