HEPATIC BIOSYNTHESIS OF CERULOPLASMIN

该建议的目的是确定细胞内位点， 过程中涉及的生物合成纳入铜， 将肝细胞转化为分泌性糖蛋白铜蓝蛋白。 的 铜掺入的细胞内位点将通过 鉴定糖蛋白加工的最早阶段， 可以检测全血浆铜蓝蛋白。 利用HepG2作为模型， 全铜蓝蛋白的合成，铜蓝蛋白在其各个阶段， 糖蛋白加工将被分离并通过糖苷酶分析 处理和凝集素亲和层析，以及HPLC分析 寡糖结构 形成的过程 将通过检查结构差异来研究血浆脱铜蓝蛋白， 原发性和继发性，分离自人的apoceruloplasmin 血清，由HepG2分泌，并从人工制备的 全蛋白 这将在小学和中学完成。 蛋白质结构的肽图谱，和氨基酸修饰的天然 蛋白质，然后肽图谱。 凝集素亲和层析和 寡肽的HPLC分析将用于检查 寡糖结构 寡糖添加抑制剂 蛋白质和糖蛋白加工酶的抑制剂， 检查了它们对铜掺入铜蓝蛋白的影响 关于HepG2 这种处理是有意义的，因为它可能导致修改 糖蛋白二级结构的变化及其 细胞内通过特定的细胞器，这可能是至关重要的 所以铜要适当地结合。 血浆铜蓝蛋白的翻译 将使用无细胞系统来确定细胞的细胞特异性。 铜结合的过程，并提供一种识别和 评价这一进程的基本组成部分的作用， 分析这些成分的变化，它们的表达和功能， 在肝脏发育过程中铜参与的细胞内过程 掺入到该蛋白质中是未知的。 占据 了解这些过程，它们的组成部分和控制 元素，将直接关系到微量金属的基本问题 健康和疾病的代谢，以及蛋白质的基本机制 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.