Purification of a sheep liver cytochrome P-450 from the P450IIIA gene subfamily. Its contribution to the N-dealkylation of veterinary drugs.

Oral administration of troleandomycin at a dose of 100 mg/kg/day for 6 days to three adult male Lacaune sheep produced a 1.6-fold increase in specific content of liver microsomal cytochrome P-450. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, microsomal preparations from treated animals exhibited a strong band in the zone of electrophoretic mobility of cytochromes P-450. This band corresponded to a cytochrome P-450 which cross-reacted with rabbit P450IIIA6 antibodies, as demonstrated by immunoblotting. The ovine isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, CM cellulose and hydroxylapatite chromatographic separations. This hemoprotein had an apparent molecular weight of 52 kD as determined by calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was characterized in terms of spectral data, NH2-terminal amino acid sequence, immunologic and catalytic properties. This study revealed some interspecies differences with the orthologous rabbit isozyme. The contribution of this form to the N-demethylation of erythromycin and of three veterinary drugs: chlorpromazine, chlorpheniramine and bromhexine was demonstrated from inhibition by TAO, from immunoinhibition studies, using polyclonal antibodies raised in rabbit and from the existence of significant correlations between its microsomal level and these N-demethylase activities. In contrast, the results suggest that ovine P450IIIA could not be predominantly involved in the N-dealkylation of benzphetamine, ephedrine, ivermectine or spiramycin.