Neskovic N M, Roussel G, Nussbaum J L
J Neurochem. 1986 Nov;47(5):1412-8. doi: 10.1111/j.1471-4159.1986.tb00773.x.
A new method for purification of UDPgalactose:ceramide galactosyltransferase (EC 2.4.1.45) is described. The principal steps involved solvent extraction at -70 degrees C, Triton X-100 extraction, and DEAE-Sephadex and Blue Sepharose chromatography. The active configuration of the enzyme was stabilized by phospholipids and a rapid loss of enzymatic activity was observed after removal of these lipids. The inactive enzyme could be fully reactivated in the presence of brain phospholipids dispersed in a Triton X-100-containing buffer. The purified enzyme preparation showed two major components by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with apparent molecular weights of 50-70,000. The 53,000-dalton protein was isolated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate and used to produce antibodies against UDPgalactose:ceramide galactosyltransferase.
本文描述了一种纯化UDP半乳糖：神经酰胺半乳糖基转移酶（EC 2.4.1.45）的新方法。主要步骤包括在-70℃下进行溶剂萃取、Triton X-100萃取以及DEAE-葡聚糖凝胶和蓝色琼脂糖凝胶色谱法。该酶的活性构型由磷脂稳定，去除这些脂质后观察到酶活性迅速丧失。在分散于含Triton X-100缓冲液中的脑磷脂存在下，无活性的酶可完全重新激活。在十二烷基硫酸钠存在下，通过聚丙烯酰胺凝胶电泳，纯化的酶制剂显示出两个主要成分，表观分子量为50 - 70,000。在十二烷基硫酸钠存在下，通过制备性凝胶电泳分离出53,000道尔顿的蛋白质，并用于制备抗UDP半乳糖：神经酰胺半乳糖基转移酶的抗体。
