Characterization of eprinomectin N-deacetylase in rats

The enzyme system responsible for the N-deacetylation of eprinomectin in rats was characterized. Tissue and subcellular studies showed that the hydrolysis activity was localized mainly in liver microsomes. Apparent KM and Vmax values calculated from Lineweaver-Burk plots were 53 microM and 0.81 nmol/mg/min for male rats and 70 microM and 4.99 nmol/mg/min for female rats, respectively. Pretreatment of male rats with dexamethasone, phenobarbital, and pregnenolone 16alpha-carbonitrile increased the activity by more than 3-fold. Paraoxon and bis-4-nitrophenylphosphate strongly inhibited the deacetylase activity at concentrations as low as 1 microM. The hydrolysis activity also was inhibited by SKF525, but less effectively. Eserine strongly inhibited the activity at 1 x 10(-4) M. HgCl2 decreased the activity to about 40% at a concentration of 1 x 10(-4) M. FeCl3, CaCl2, MgCl2, and EDTA had little effect on the hydrolysis of eprinomectin, whereas NaF slightly increased the activity to 118%. Thus, the inhibition study suggested that eprinomectin deacetylase resembled "B" type carboxylesterase/amidases. The hydrolysis activity of eprinomectin and isocarboxazid, a specific substrate of RL2 [Hosokawa, M, Maki T and Satoh T (1987) Mol Pharmacol 31:579-584], by liver microsomes from rats treated with various cytochrome P-450 inducers correlated well (r = 0.92). Also, elusion profiles of esterase by gel filtration and ion exchange chromatography demonstrated that the active protein(s) for eprinomectin and isocarboxazid hydrolysis coeluted. Thus, RL2 or an enzyme system similar to RL2 is responsible for the N-deacetylation of eprinomectin.