Fleischer B
J Cell Biol. 1981 May;89(2):246-55. doi: 10.1083/jcb.89.2.246.
UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP-sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.
大鼠肝脏高尔基体的UDP-半乳糖：N-乙酰葡糖胺半乳糖基转移酶（GT）和CMP-唾液酸：去唾液酸转铁蛋白唾液酸转移酶（ST）活性是膜结合酶，可用Triton X-100处理将其释放。当使用蛋白质底物在新制备的高尔基体囊泡中测定这些酶时，加入Triton X-100后两种活性均增强约8倍。然而，当使用小分子量底物时，加入去污剂后两种活性仅增强约2倍。即使将高尔基体制剂在液氮中冷冻保存2个月后，大蛋白质底物仍无法接触到这些酶。经过这样的保存后，对小分子量底物的可及性显著增加。高尔基体囊泡中的GT和ST活性不会因胰蛋白酶处理而被破坏，但如果囊泡先用Triton X-100破坏，则会被这种处理破坏。用低水平的制霉菌素（一种已知可与生物膜中的胆固醇结合的多烯抗生素）处理高尔基体囊泡，也会导致两种糖基转移酶对胰蛋白酶的敏感性增强。当制霉菌素与总胆固醇的重量比约为1.6或摩尔比约为1时，胰蛋白酶对糖基转移酶活性的破坏最大。这个制霉菌素水平不会使酶溶解，但会导致高尔基体膜出现褶皱（通过电子显微镜可见），并导致高尔基体囊泡破裂（通过血清白蛋白的释放来衡量）。当分离的高尔基体用戊二醛固定以维持潴泡和分泌囊泡的三维取向，然后用制霉菌素处理时，高尔基体顺面和反面的潴泡膜以及分泌颗粒膜似乎受到的影响大致相同。这些结果表明，分离出的肝脏高尔基体囊泡在很大程度上是将GT和ST定位在膜的腔面，这与肝细胞中高尔基体的潴泡区室相对应。胆固醇是高尔基体膜的一个组成部分，其在整个高尔基体中的分布与两种转移酶的分布相似。
