GLYCOSYLATION MECHANISMS OF CELL LIPIDS AND PROTEINS

长期的目标是了解调控细胞的细胞机制 粗面内质网中脂质和蛋白质的糖基化 (RER)和高尔基体(GA)。我们建议：(1)继续学习 糖基化在GA中的作用机制。我们将尝试确认， 提纯假定的糖核苷酸并将其重组为脂质体 大鼠GA膜转位蛋白的研究进展 肝脏。具有光反应基团的特定放射性标记的糖核苷酸 在核苷酸部分中将使用。这些研究也将进行 用来自野生型的高尔基体小泡和CMP-唾液酸转运阴性 近年来，UDP-半乳糖转运阴性中国仓鼠卵巢细胞 这是我们实验室的特点。针对这些转运体的抗体将是 获得并用于在体内定位GA内的转运子和 以确定移位器和对应的 GA膜上的糖基转移酶。(2)继续研究 粗面内质网中糖基化的机制。Rough的脉冲追逐实验 将进行微粒子和放射性糖核苷酸检测以确定 这些囊泡中糖基化的脂寡糖的形态。我们 还应尝试鉴定、提纯和重组UDP-葡萄糖 和UDP-GlcNAc将RER转运蛋白转移到脂质体中 类似于为GA描述的那些。(3)进一步具体化 CMP-唾液酸合成酶，一种核质酶。我们将完成 酶的纯化及多克隆和单抗的制备 与合成酶比对。这些将被用来研究合成的地点 以及酶在细胞内和长期研究中的定位 试图了解这些蛋白质是如何迁移到细胞核的。(4) 选择性获得大鼠肝高尔基体囊泡亚群的尝试 富含不同的高尔基体酶活性。核苷酸和抗体 抗已偶联到琼脂糖凝胶上的糖核苷酸转运体 将会被使用。如果成功，这些囊泡可能会被用来研究 糖蛋白在GA内的运输。(五)继续保持优势互补 上述体外研究试图通过分离和鉴定突变体 中国仓鼠卵巢细胞缺乏相应的反应 活着。曾遭受过放射性自杀的细胞 氚唾液酸和岩藻糖将通过复制品进行突变筛查 电镀放射自显影，然后是生化和遗传学 互补性分析。

The long-term goal is to understand the cellular mechanisms that regulate the glycosylation of lipids and proteins in the rough endoplasmic reticulum (RER) and Golgi apparatus (GA). We propose to: (1) Continue our studies on the mechanism of glycosylation in the GA. We will attempt to identify, purify, and reconstitute into liposomes the putative sugar nucleotide translocator proteins recently characterized in the GA membrane from rat liver. Specific radiolabeled sugar nucleotides with photoreactive groups in the nucleotide moieties will be used. These studies will also be done with Golgi vesicles from wild-type and CMP-sialic acid transport negative and UDP-Galactose transport negative Chinese hamster ovary cells recently characterized in our lab. Antibodies against these translocators will be obtained and used to localize the translocators within the GA in vivo and to determine the relationship between the translocators and corresponding glycosyltransferases in the GA membrane. (2) Continue our studies on the mechanism of glycosylation in the RER. Pulse-chase experiments with rough microsomes and radioactive sugar nucleotides will be done to determine the topography of glycosylated lipid oligosaccharides in these vesicles. We shall also attempt to identify, purify, and reconstitute the UDP-Glucose and UDP-GlcNAc translocators of the RER into liposomes using approaches similar to those described for the GA. (3) Further characterize CMP-sialic acid synthetase, a nucleoplasmic enzyme. We shall complete the purification of the enzyme and prepare polyclonal and monoclonal antibodies against the synthetase. These will be used to study the site of synthesis and localization of the enzyme within the cell and in longer-term studies to attempt to understand how such proteins migrate to the nucleus. (4) Attempt to obtain subpopulations of rat liver Golgi vesicles selectively enriched in different Golgi enzyme activities. Nucleotides and antibodies against sugar nucleotide translocators that have been coupled to Sepharose will be used. If successful, these vesicles may be of use to study the transport of glycoproteins within the GA. (5) Continue to complement the above studies in vitro by attempting to isolate and characterize mutant Chinese hamster ovary cells deficient in the corresponding reactions in vivo. Cells which have been subjected to radioactive suicide with tritiated sialic acid and fucose will be screened for mutations via replica plating autoradiography, followed by biochemical and genetic complementation analyses.