Gonatas J O, Mezitis S G, Stieber A, Fleischer B, Gonatas N K
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia.
J Biol Chem. 1989 Jan 5;264(1):646-53.
A monoclonal antibody (mAb 10A8), derived from mice immunized with fractions of the Golgi apparatus from rat brain neurons, was exploited to isolate and partially characterize a novel glycoprotein of 160 kDa apparent molecular mass which was localized by immunoelectron microscopy in medial cisternae of the Golgi apparatus of neurons, glia, pituitary cells, and rat pheochromocytoma (PC 12). The yield of immunoaffinity purified protein was 0.9 microgram/g of rat brain and represented 3% of the Golgi protein; the protein contained asparagine-linked carbohydrates and sialic acid and N-acetylglucosamine residues; unreduced protein had a greater electrophoretic mobility (130 kDa) consistent with the presence of intrachain disulfide bonds. The bulk of the glycoprotein resided within the membrane and/or luminal face of the Golgi cisternae. After extraction with Triton X-114, the glycoprotein was found in both aqueous and detergent phases. The monoclonal antibody did not inhibit the activities of Golgi enzymes or the uptake of nucleotide sugars by intact Golgi vesicles. The findings indicate that the 160-kDa glycoprotein is a specific constituent of medial Golgi cisternae. The results of this study lend support to the hypothesis that the distributions of glycosyltransferases in the Golgi apparatus are cell specific, since in neurons this sialic acid containing glycoprotein is found in medial rather than in trans and/or in the trans Golgi reticulum cisternae, where sialyltransferases have been localized in other cells. Alternatively, resident neuronal Golgi sialoglycoproteins may acquire sialic acid in trans elements of the apparatus and then shuttle back in medial cisternae.
一种单克隆抗体（mAb 10A8），由用大鼠脑神经元高尔基体部分免疫的小鼠产生，用于分离和部分表征一种表观分子量为160 kDa的新型糖蛋白，通过免疫电子显微镜定位在神经元、神经胶质细胞、垂体细胞和大鼠嗜铬细胞瘤（PC 12）的高尔基体中间潴泡中。免疫亲和纯化蛋白的产量为0.9微克/克大鼠脑，占高尔基体蛋白的3%；该蛋白含有天冬酰胺连接的碳水化合物以及唾液酸和N - 乙酰葡糖胺残基；未还原的蛋白具有更大的电泳迁移率（130 kDa），这与链内二硫键的存在一致。大部分糖蛋白位于高尔基体潴泡的膜和/或腔内表面。用Triton X - 114提取后，糖蛋白在水相和去污剂相中均被发现。单克隆抗体不抑制高尔基体酶的活性或完整高尔基体囊泡对核苷酸糖的摄取。这些发现表明160 kDa糖蛋白是高尔基体中间潴泡的一种特异性成分。本研究结果支持了高尔基体中糖基转移酶的分布具有细胞特异性这一假说，因为在神经元中，这种含唾液酸的糖蛋白存在于中间潴泡而非反式和/或反式高尔基体网状潴泡中，而在其他细胞中唾液酸转移酶已定位在反式和/或反式高尔基体网状潴泡中。或者，神经元高尔基体驻留唾液酸糖蛋白可能在高尔基体的反式元件中获得唾液酸，然后再回到中间潴泡中。
