Strous G J, Van Kerkhof P, Willemsen R, Geuze H J, Berger E G
J Cell Biol. 1983 Sep;97(3):723-7. doi: 10.1083/jcb.97.3.723.
HeLa cell membranes were studied for the distribution and orientation of the Golgi marker enzyme uridine diphosphate-galactose:beta-D-N-acetylglucosamine beta, 1-4 transferase (GT). Short pulse labeling in the presence of [35S]methionine resulted in two precursor species (Mr = 44,000 and 47,000), present in a microsomal fraction with a density of 1.18 g/ml in sucrose, presumably derived from the rough endoplasmic reticulum. Processing of the N-linked oligosaccharide(s) occurred only after the precursor molecules migrated to lighter density fractions, presumably derived from the Golgi complex. The mature GT molecules (Mr = 54,000) contain O-linked oligosaccharides as shown by beta-elimination of metabolically incorporated [3H]galactose. The O-glycosylation occurred mainly in the light density fractions. The topology of GT was studied on membrane fractions after labeling with [35S]methionine as well as immunocytochemically on ultrathin cryosections at the electron microscope level. Our results indicate that both the antigenic determinants of GT as well as polypeptide chain are present intramembraneously and at the luminal side of the membranes of the Golgi complex and rough endoplasmic reticulum.
对HeLa细胞膜进行了研究，以确定高尔基体标记酶尿苷二磷酸半乳糖：β-D-N-乙酰葡糖胺β，1-4转移酶（GT）的分布和方向。在[35S]甲硫氨酸存在下进行短脉冲标记，产生了两种前体物质（分子量分别为44,000和47,000），存在于蔗糖密度为1.18 g/ml的微粒体部分，推测来源于糙面内质网。只有在前体分子迁移到密度较轻的部分（推测来源于高尔基体复合体）后，N-连接寡糖的加工才会发生。成熟的GT分子（分子量为54,000）含有O-连接寡糖，这通过对代谢掺入的[3H]半乳糖进行β-消除得以证明。O-糖基化主要发生在低密度部分。在用[35S]甲硫氨酸标记后的膜部分以及在电子显微镜水平对超薄冰冻切片进行免疫细胞化学研究中，对GT的拓扑结构进行了研究。我们的结果表明，GT的抗原决定簇以及多肽链都存在于膜内以及高尔基体复合体和糙面内质网的膜腔侧。
