Tsujita T, Miyazaki T, Tabei R, Okuda H
Department of Medical Biochemistry, School of Medicine, Ehime University, Ehime, Japan.
J Biol Chem. 1996 Jan 26;271(4):2156-61. doi: 10.1074/jbc.271.4.2156.
Rat intestinal mucosa contains high diacylglycerol-synthesizing activity (monoacylglycerol acyltransferase (MGAT) activity) due to monoacylglycerol and fatty acid, independently of coenzyme A and ATP. MGAT activity was purified from rat intestinal mucosa by successive chromatography separations on DEAE-cellulose, CM- Sephadex, and anti-IgG-Sepharose against rat pancreatic lipase. The enzyme was electrophoretically homogeneous, and its molecular weight was 49,000, which is identical with that of rat pancreatic lipase. Immunoblotting analysis with antibody against rat pancreatic lipase showed one immunoreactive protein with an estimated molecular weight of 49,000. The activity of the purified enzyme was completely inhibited by addition of the antibody. Using immunocytochemical techniques, it was found that immunoreactive protein against rat pancreatic lipase was uniformly distributed within the absorptive cells of the intestine but was absent from the microvillar membrane. The MGAT activity of intestinal mucosal homogenate was inhibited by about 65% by addition of antibody against rat pancreatic lipase. Trioleoylglycerol- and dioleoylglycerol-hydrolyzing activities of the purified enzyme and pancreatic lipase were inhibited by addition of intestinal mucosa extract. These results suggest that pancreatic lipase is present in intestinal absorptive cells and that it may contribute to resynthesis of diacylglycerol from monoacylglycerol and fatty acids in these cells.
大鼠肠黏膜含有较高的二酰甘油合成活性（单酰甘油酰基转移酶（MGAT）活性），该活性由单酰甘油和脂肪酸产生，与辅酶A和ATP无关。通过在DEAE-纤维素、CM-葡聚糖凝胶以及抗大鼠胰脂肪酶的抗IgG-琼脂糖凝胶上进行连续色谱分离，从大鼠肠黏膜中纯化出MGAT活性。该酶在电泳上是均一的，其分子量为49,000，与大鼠胰脂肪酶的分子量相同。用抗大鼠胰脂肪酶抗体进行免疫印迹分析显示，有一个估计分子量为49,000的免疫反应性蛋白。加入抗体后，纯化酶的活性被完全抑制。利用免疫细胞化学技术发现，抗大鼠胰脂肪酶的免疫反应性蛋白在肠吸收细胞内均匀分布，但在微绒毛膜中不存在。加入抗大鼠胰脂肪酶抗体后，肠黏膜匀浆的MGAT活性被抑制约65%。加入肠黏膜提取物后，纯化酶和胰脂肪酶的三油酰甘油和二油酰甘油水解活性受到抑制。这些结果表明，胰脂肪酶存在于肠吸收细胞中，并且它可能有助于这些细胞中单酰甘油和脂肪酸重新合成二酰甘油。
