SOLUBILIZATION AND PURIFICATION OF A-ESTERASE FROM MOUSE HEPATIC MICROSOMES

A-esterase(s), an enzyme(s) that hydrolyzes certain organophosphate co mpounds, is located in mammals, primarily in serum and liver. Although considerable information is available regarding serum A-esterase(s), little is known about the hepatic form(s) of this enzyme. In the prese nt study, hepatic A-esterase activity was quantified by measuring the EDTA-sensitive hydrolysis of the organophosphate paraoxon (O,O-diethyl -O-p-nitrophenyl phosphate). EDTA-insensitive hydrolysis was assumed t o be the nonenzymatic phosphorylation of proteins with appropriate ser ine hydroxyl groups. Resuspension of mouse hepatic microsomes in 50 mM potassium phosphate buffer, pH 7.4, containing 100 mu M calcium chlor ide, 0.25% sodium cholate, and 0.1% Triton N-101, resulted in the solu bilization of A-esterase activity, as evidenced by the failure of acti vity to sediment after centrifugation at 100,000 g for 1 hr. Gel perme ation chromatography followed by ion-exchange chromatography and nonsp ecific affinity chromatography resulted in a peak of A-esterase activi ty judged to be homogeneous by SDS-PAGE. A typical purification result ed in a 1531-fold increase in specific activity, with a recovery of 10 %. SDS-PAGE with and without an acrylamide gradient indicated a molecu lar weight of 40,000 and 39,000 Da, respectively, while analyses of am ino acid composition revealed similarities with human and rabbit serum paraoxonase. And finally, although this protein hydrolyzed both parao xon and methyl paraoxon (O, O-dimethyl-O-p-nitrophenyl phosphate), it did not hydrolyze p-nitrophenyl acetate.