HEPATIC BIOSYNTHESIS OF CERULOPLASMIN

这项建议的目标是确定细胞内位置和 铜的生物合成过程中涉及的过程 肝细胞转化为分泌型糖蛋白铜蓝蛋白。这个 铜掺入的胞内位置将通过以下方式确定 糖蛋白加工的最早阶段的鉴定 可检测到全血清蓝蛋白。利用HepG2作为模型 全铜蓝蛋白、铜蓝蛋白在ITS不同阶段的合成 糖苷酶对糖蛋白加工进行分离和分析 处理和凝集素亲和层析以及高效液相色谱分析 低聚糖结构。形成过程中所涉及的过程 将通过检查结构差异来研究载脂蛋白， 从人类分离的载角蛋白原和继发性之间 血清，由HepG2分泌，以及人工从 全蛋白。这将在主服务器和辅助服务器上完成 多肽图谱的蛋白质结构和天然氨基酸的修饰 蛋白质，然后是多肽图谱。凝集素亲和层析和 低聚肽的高效液相色谱分析将被用来检验 低聚糖结构。低聚糖加成的抑制剂 蛋白质和糖蛋白加工酶的抑制物，将是 考察它们对铜蓝蛋白掺入铜的影响 由赫普盖尔写的。这种处理是有意义的，因为它可能会导致修改 糖蛋白的二级结构及其变化 通过特定细胞器的细胞内通道，这可能是至关重要的 将铜适当地结合起来。铜蓝蛋白在日本的翻译 无细胞系统将被用来确定细胞特异性 铜结合过程，并提供了一种识别和 评价这一进程的基本组成部分的作用，以及 分析这些成分的变化，它们的表达和功能， 肝脏发育过程中与铜有关的细胞内过程 到目前为止，这种蛋白质的结合情况尚不清楚。获得了一个 了解这些过程、它们的组成部分及其控制 元素，将直接关系到痕量金属的基本问题 健康与疾病中的代谢以及蛋白质的基本机制 正在处理。

The objective of this proposal is to determine the intracellular site and processes involved in the biosynthetic incorporation of copper by hepatocytes into the secretory glycoprotein, ceruloplasmin. The intracellular site of copper incorporation will be determined by identification of the earliest stage of glycoprotein processing that holoceruloplasmin may be detected. Utilizing HepG2 as a model for the synthesis of holoceruloplasmin, ceruloplasmin in the various stages of its glycoprotein processing will be isolated and analyzed by glycosidase treatment and lectin affinity chromatography, as well as HPLC analysis of oligosaccharide structure. The processes involved in the formation of apoceruloplasmin will be studied by examining for structural differences, both primary and secondary, between apoceruloplasmin isolated from human serum, secreted by HepG2, and that prepared artificially from the holoprotein. This will be accomplished for the primary and secondary protein structure by peptide mapping, and amino acid modification of native proteins followed by peptide mapping. Lectin affinity chromatography and HPLC analysis of oligopeptides will be used to examine differences in oligosaccharide structure. Inhibitors of oligosaccharide addition to proteins, and inhibitors of glycoprotein processing enzymes, will be examined for their effect on the incorporation of copper into ceruloplasmin by HepG2. Such treatment is of interest as it may result in modification of the glycoproteins secondary structure and alteration of its intracellular passage through particular organelles, which may be critical fore copper to incorporate appropriately. Translation of ceruloplasmin in a cell-free system will be used to determine the cell specificity of the proces of copper incorporation, and provide a means for identification and evaluation of the role of essential components of this process, and analysis of the changes in these components, their expression and function, during hepatic development The intracellular processes involved in copper incorporation into this protein are as yet unknown. Gaining an understanding of these processes, their components and their controlling elements, will bear directly upon fundamental problems of trace metal metabolism in health and disease, and upon basic mechanisms of protein processing.