David_1998_Eur.J.Biochem_257_142

A glycerol-ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight-step purification procedure. The isolation scheme used gave a 483-fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 micromol x min(-1) x mg(-1). The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size-exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N-terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full-length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol-ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97% identity with that of porcine liver carboxylesterase and more than 50% identity with those of other carboxylesterases from different mammalian species.