GLYCOSYLATION MECHANISMS OF CELL LIPIDS AND PROTEINS

长期的目标是了解调节细胞的细胞机制， 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）继续我们的研究 GA中糖基化的机制。 我们将尝试识别， 纯化并将推定的糖核苷酸重组到脂质体中 最近在大鼠GA膜上鉴定的转运蛋白 肝脏 具有光反应性基团的特异性放射性标记的糖核苷酸 在核苷酸部分中使用。 这些研究也将在 与野生型和CMP-唾液酸转运阴性的高尔基体囊泡 和UDP-半乳糖转运阴性的中国仓鼠卵巢细胞 在我们的实验室里。 针对这些转运子的抗体将被 获得并用于在体内定位GA内的易位子， 以确定易位体和相应的 GA膜中的糖基转移酶。 (2)继续研究 RER中糖基化的机制。 粗糙集脉冲追踪实验 微粒体和放射性糖核苷酸将被用来确定 这些囊泡中糖基化的脂质寡糖的拓扑结构。 我们 还应尝试识别、纯化和重组UDP-葡萄糖 和RER的UDP-GlcNAc易位体进入脂质体， 类似于对GA所描述的那些。 (3)进一步表征 CMP-唾液酸合成酶，核质酶。 我们将完成 纯化酶并制备多克隆和单克隆抗体 对抗合成酶 这些将用于研究合成的位点 以及酶在细胞内的定位， 试图了解这些蛋白质是如何迁移到细胞核的。 （四） 选择性获得大鼠肝高尔基体泡亚群的尝试 富含不同的高尔基体酶活性。 核苷酸和抗体 针对已经偶联到琼脂糖凝胶上的糖核苷酸移位器， 将用于 如果成功的话，这些囊泡可能有助于研究 糖蛋白在GA内的转运。 (5)继续补充 通过尝试分离和表征突变体， 中国仓鼠卵巢细胞缺乏相应的反应， vivo. 细胞已经受到放射性自杀， 氚化唾液酸和岩藻糖将通过复制品筛选突变 平板放射自显影，然后进行生化和遗传学检查。 互补分析

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.