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.