David L, Guo X J, Villard C, Moulin A, Puigserver A
Laboratoire de Biochimie et Biologie de la Nutrition, CNRS-ESA 6033, Faculté des Sciences et Techniques St-Jérôme, Marseille, France.
Eur J Biochem. 1998 Oct 1;257(1):142-8. doi: 10.1046/j.1432-1327.1998.2570142.x.
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.
采用部分脱脂法和八步纯化程序，从猪小肠黏膜中纯化出一种甘油酯水解酶，使其达到同质。所采用的分离方案实现了483倍的纯化，得到了一种对三丁酸甘油酯具有290微摩尔·分钟⁻¹·毫克⁻¹比活性的纯酶。根据尺寸排阻色谱结果，该酶的分子量估计为240 kDa，而通过SDS/PAGE分析确定为60 kDa。所获得的等电聚焦数据表明，仅存在一种等电点为5.1的同工型。发现该酶前25个氨基酸残基的N端序列与猪肝羧酸酯酶的N端序列完全相同。从猪小肠中分离出编码该酶的全长cDNA。我们观察到，最初命名为肠甘油酯水解酶的相应蛋白质明确属于羧酸酯酶家族。推导的氨基酸序列由565个残基组成，与猪肝羧酸酯酶的氨基酸序列具有97%的同一性，与来自不同哺乳动物物种的其他羧酸酯酶的氨基酸序列具有超过50%的同一性。
