PURIFICATION OF 2 RAT HEPATIC PROTEINS WITH A-ESTERASE ACTIVITY TOWARD CHLORPYRIFOS-OXON AND PARAOXON

A-esterases are calcium-dependent hydrolases that can detoxify the act ive metabolites (oxons) of organophosphorus insecticides such as chlor pyrifos and parathion. A-esterases from rat liver have previously been shown to hydrolyze chlorpyrifos-oxon but not paraoxon at low substrat e concentrations. Two A-esterases were extracted by ammonium sulfate f ractionation from solubilized rat liver microsomes followed by gel fil tration chromatography and preparative scale isoelectric focusing. The proteins displayed similar characteristics and were difficult to sepa rate; both had similar high molecular mass and isoelectric point range and exhibited A-esterase activity toward high and low concentrations of chlorpyrifos-oxon and high concentrations of paraoxon. Sufficient a mounts of the higher molecular mass protein were obtained for kinetic studies, which yielded a K-m of 0.93 mM toward high concentrations of chlorpyrifos-oxon and a V-max of 369 nmoles product formed/mg protein- min. The protein hydrolyzed phenyl acetate, chlorpyrifos-oxon and para oxon, suggesting that arylesterase and A-esterase activities are attri butable to the same liver protein(s). Assays of purified protein and k inetic studies of microsomes suggested that the activity toward high ( 320 mu M) and low (less than or equal to 10(-5) M) concentrations of c hlorpyrifos-oxon are due to the same protein(s), and that the activity toward low concentrations of chlorpyrifos-oxon is attributable to bot h a higher affinity and a higher V-max (but primarily the latter) for chlorpyrifos-oxon than for paraoxon, which is not detectably hydrolyze d at low concentrations. The higher A-esterase activity with chlorpyri fos-oxon than paraoxon may be a major determinant in the observed lowe r acute toxicity of chlorpyrifos than parathion.