Solubilization and purification of A-esterase from mouse hepatic microsomes.
A-esterase(s), an enzyme(s) that hydrolyzes certain organophosphate compounds, 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 present 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 to be the nonenzymatic phosphorylation of proteins with appropriate serine hydroxyl groups. Resuspension of mouse hepatic microsomes in 50 mM potassium phosphate buffer, pH 7.4, containing 100 microM calcium chloride, 0.25% sodium cholate, and 0.1% Triton N-101, resulted in the solubilization of A-esterase activity, as evidenced by the failure of activity to sediment after centrifugation at 100,000 g for 1 hr. Gel permeation chromatography followed by ion-exchange chromatography and nonspecific affinity chromatography resulted in a peak of A-esterase activity judged to be homogeneous by SDS-PAGE. A typical purification resulted in a 1531-fold increase in specific activity, with a recovery of 10%. SDS-PAGE with and without an acrylamide gradient indicated a molecular weight of 40,000 and 39,000 Da, respectively, while analyses of amino acid composition revealed similarities with human and rabbit serum paraoxonase. And finally, although this protein hydrolyzed both paraoxon and methyl paraoxon (O,O-dimethyl-O-p-nitrophenyl phosphate), it did not hydrolyze p-nitrophenyl acetate.
Huang
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YS
Department of Pharmacology and Toxicology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
Woods
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Sultatos
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification*; D005260:Female; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D010261:Paraoxon / Q000031:analogs & derivatives / Q000378:metabolism; D010477:Perfusion
D000596:Amino Acids; D004950:Esterases; D010261:Paraoxon; C008480:methylparaoxon