Purification and Characterization of a Carboxylesterase from Rabbit Liver Lysosomes

Abstract

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 diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10^-4M, but was not affected by eserine, or by α- or β-naphthyl acetate at 10_-3M. Various metal ions (Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Cu²⁺, Zn²⁺, Ni²⁺) at 10^-3M also had no effect on the enzyme activity.