Purification and characterization of a carboxylesterase from rabbit liver lysosomes.
Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.
D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002850:Chromatography, Gel; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007531:Isoflurophate / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009284:Naphthols / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010830:Physostigmine / Q000494:pharmacology; D011817:Rabbits; D012995:Solubility; D013379:Substrate Specificity
D005227:Fatty Acids; D008670:Metals; D009284:Naphthols; D007531:Isoflurophate; C005366:naphthyl acetate; D010664:Phenylmethylsulfonyl Fluoride; D010830:Physostigmine; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase