title: Purification, biochemical characterization, and biological function of human esterase D.
abstract: Human esterase D (carboxylesterase; carboxylic-ester hydrolase, EC 3.1.1.1), a genetic marker of retinoblastoma, was purified to biochemical homogeneity from erythrocytes. The purification scheme including carboxymethylcellulose, phenyl-Sepharose, chromatofocusing, and hydroxylapatite chromatographies resulted in a 10,000-fold purification of the enzyme with 15% recovery of total activity. The Km of esterase D was estimated to be 10 X 10(-6) M using 4-methylumbelliferyl acetate as substrate. The enzymatic activity was inhibited by p-chloromercuribenzoate and HgCl2, suggesting an important role of SH group(s) in enzyme function. Specific rabbit polyclonal and mouse monoclonal antibodies against esterase D were prepared and recognized either denatured or native human esterase D protein. Moreover, the polyclonal antibodies immunoprecipitated a polypeptide with a molecular mass of about 33-34 kDa from various cell lines of different mammalian species, indicating that the esterase D protein is highly conserved. The highest levels of this enzyme were found in liver and kidney. Furthermore, the expression of esterase D was enhanced 3-fold in a promonocytic cell line treated with phenobarbital but not with phorbol myristate acetate, suggesting that esterase D may have a role in detoxification. The availability of the homogeneous protein and its specific antibodies allows for cloning of the esterase D gene and facilitates studies of retinoblastomas.
authors.forename: W H
authors.initials: WH
authors.lastname: Lee
authors.forename: W
authors.initials: W
authors.lastname: Wheatley
authors.forename: W F
authors.initials: WF
authors.lastname: Benedict
authors.forename: C M
authors.initials: CM
authors.lastname: Huang
authors.forename: E Y
authors.initials: EY
authors.lastname: Lee
mesh_terms: D000906:Antibodies / Q000276:immunology; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification*; D004790:Enzyme Induction; D006801:Humans; D007700:Kinetics; D010634:Phenobarbital / Q000494:pharmacology
chemical_list: D000906:Antibodies; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C020118:ESD protein, human; D010634:Phenobarbital