Studies on Glycolipid Transfer Protein

从猪脑中纯化的糖脂转移蛋白（GL-TP）含有表观分子量为22K的主要成分（80%）和表观分子量为21K的次要成分（15%）。主成分每个分子有3个游离巯基而次成分每个分子有1个游离巯基。两个组分共有的一个巯基位于分子表面，可以用正乙基马来酰亚胺修饰。当其他两个巯基形成分子内二硫键时，次要组分由主要组分形成。目前得到的结果表明，具有分子内二硫键的GL-TP的活性比还原形式的GL-TP高两倍。发现GL-TP形成GL-TP的比例更大。糖脂复合物，转移反应的中间产物，当GL-TP含有分子内二硫键时。纯化后的GL-TP用溴化氰进行裂解。从裂解形成的多肽中确定部分氨基酸序列。基于获得的序列，cDNA克隆正在进行中。制备了针对纯化的GL-TP的特异性抗体。在猪上皮细胞系PK（15）细胞、ret脑、人肾、小鼠神经母细胞瘤细胞系和小鼠嗜铬细胞瘤细胞系PC12细胞中发现了一种分子量约为20K的蛋白，与抗体产生免疫反应，并具有糖脂转移活性。GL-TP似乎存在于大多数动物细胞中。对GL-TP在PK（15）细胞亚细胞定位的研究表明，GL-TP是一种细胞质蛋白。GL-TP似乎参与了在顺式高尔基膜细胞质侧合成的葡萄糖神经酰胺到中间和反式高尔基池的易位，在那里，葡萄糖神经酰胺的进一步糖基化发生在高尔基体的管腔表面。

Glycolipid transfer protein (GL-TP) purified from pig brain contains the major component (80%) of apparent molecular weight of 22K and the minor component (15%) of molecular weight of 21K. The major component has three free sulfhydryl groups per molecule and the minor component has one free sulfhydryl group per molecule. One sulfhydryl group common to both components locates on the surface of the molecule and can be modified with n-ethylmaleimide. The minor component is formed from the major component when an intramolecular disulfide bond is formed from the other two sulfhydryl groups. Results so far obtained indicate that the GL-TP with an intramolecular disulfide bond has twice more activity than the reduced form of GL-TP. Much larger partion of GL-TP was found to form GL-TP. Glycolipid complex, an intermediate of the transfer reaction, when GL-TP contained an intramolecular disulfide bond.Purified GL-TP was subjected to the cleavage with cyanogen bromide. Partial amino acid sequence was determined from the peptides formed by the cleavage. A cDNA cloning is in progress based on the sequence obtained.Specific antibody against the purified GL-TP was prepared. A protein of molecular weight about 20K, immunoreactive with the antibody, and with glycolipid transfer activity has been found in PK(15) cells, a porcine epithelial cell line, ret brain, human kidney, mouse neuroblastoma cell lines, and PC12 cells, a mouse pheochromocytoma line. GL-TP seems to be present in most animal cells. A study on the subcellular localization of GL-TP in PK(15) cells indicates that GL-TP is a cytoplasmic protein. GL-TP seems to participate in the transloctaion of glucosylceramide, synthesized on the cytoplasmic side of cis Golgi membrane, to middle and trans Golgi cisternae, where further glycosylation of glucosylceramide takes place on the luminal face of the Golgi apparatus.