Manganaro F, Kuksis A
Can J Biochem Cell Biol. 1985 Feb;63(2):107-14. doi: 10.1139/o85-016.
A triacylglycerol synthetase complex made up of acyl-CoA synthetase, acyl-CoA:monoacylglycerol acyltransferase, and acyl-CoA:diacylglycerol acyltransferase has been solubilized by sodium taurocholate and isolated by chromatography on phenyl-Sepharose. For this purpose microsomes of the villus cells of rat intestinal mucosa were dissolved in 2% sodium taurocholate prepared in 1 M (NH4)2SO4 and 25 mM Tris-HCl (pH 8.5) (buffer A). After dialysis against buffer A, the sample was loaded on a phenyl-Sepharose column and the enzyme complex was eluted with 25 mM Tris-HCl (pH 8.5) (buffer B). The enzymatically active fractions were pooled and rechromatographed on a Bio-Gel A-0.5m column equilibrated with buffer B and 150 mM NaCl. The recovered triacylglycerol synthetase complex accounted for over 75% of the original enzyme activity and represented a 145-fold purification from villus cells and a 10-fold purification from microsomes. It exhibited maximal activity at pH 8.0-9.0. On the basis of Bio-Gel A-0.5m chromatography the three enzymic activities appeared as a single fraction in the molecular weight range of 350 000-375 000. The complex migrated as a single peak on high performance liquid chromatography on an ion-exchange column using a NaCl gradient. The ratio of the activities of the three enzymes remained constant during the purification. The purified enzyme complex lost about 50% of its diacylglycerol acyltransferase activity on storage for 2 weeks at -20 degrees C in presence of phenylmethylsulfonyl fluoride.
一种由酰基辅酶A合成酶、酰基辅酶A：单酰甘油酰基转移酶和酰基辅酶A：二酰甘油酰基转移酶组成的三酰甘油合成酶复合物已被牛磺胆酸钠溶解，并通过在苯基琼脂糖上的色谱法分离出来。为此，将大鼠肠黏膜绒毛细胞的微粒体溶解在1 M硫酸铵和25 mM Tris-HCl（pH 8.5）（缓冲液A）中制备的2%牛磺胆酸钠中。在与缓冲液A透析后，将样品加载到苯基琼脂糖柱上，并用25 mM Tris-HCl（pH 8.5）（缓冲液B）洗脱酶复合物。将具有酶活性的部分合并，并在与缓冲液B和150 mM氯化钠平衡的Bio-Gel A-0.5m柱上重新进行色谱分析。回收的三酰甘油合成酶复合物占原始酶活性的75%以上，代表从绒毛细胞中纯化了145倍，从微粒体中纯化了10倍。它在pH 8.0 - 9.0时表现出最大活性。基于Bio-Gel A-0.5m色谱分析，这三种酶活性在分子量范围为350000 - 375000时呈现为单一部分。该复合物在使用氯化钠梯度的离子交换柱上进行高效液相色谱分析时迁移为单峰。在纯化过程中，这三种酶的活性比例保持恒定。在存在苯甲基磺酰氟的情况下，将纯化的酶复合物在-20℃下储存2周后，其二酰甘油酰基转移酶活性损失了约50%。
