Purification and molecular cloning of porcine intestinal glycerol-ester hydrolase--evidence for its identity with carboxylesterase.
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.
David
L
L
Laboratoire de Biochimie et Biologie de la Nutrition, CNRS-ESA 6033, Faculté des Sciences et Techniques St-Jérôme, Marseille, France.
Guo
X J
XJ
Villard
C
C
Moulin
A
A
Puigserver
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007422:Intestines / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008994:Monoacylglycerol Lipases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
D002241:Carbohydrates; D018076:DNA, Complementary; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D008994:Monoacylglycerol Lipases