This proposal is a continuation of a long-term study of the structure of mammalian glycogen and how this giant molecule originates in the cell. We have evidence that in rabbit muscle the glycogen is convalently bound at two points to a protein (we call the latter glycogenin) which we believe is the biosynthetic origin of the molecule, i.e. glycogen is constructed on glycogenin as a post-translational event to the synthesis of the protein. Our evidence for the linkage between glycogen and glycogenin is that it is a new type of carbohydrate to protein bond, involving tyrosine, through its hydroxyl group. Insulin, with its still unexplained ability to promote glycogen synthesis in muscle, acts on its receptor so as to cause the latter to become an active kinase which proceeds to phosphorylate itself and other cell proteins at the tyrosine hydroxyl group. The connection between tyrosine phosphorylation and the construction of glycogen on a tyrosine residue may provide a clue to how insulin stimulates glycogen synthesis. The discovery of glycogenin may also have wider utility in explaining how other storage polysaccharides originate. Another structural featrue of muscle and liver glycogen that we have defined in some detail is the occurrence of two types of phosphate ester. Some glucose residues carry a 6-phosphate group. Others carry what appears to be a phosphodiester. We will further investigate the glycogen-protein bond, seeking additional evidence for the involvement of tyrosine. Then we will determine the peptide sequences of muscle glycogenin in the regions where it is joined to glycogen and determine the precise structure of the glycogen around the linkage to protein. Similar, less-detailed studies will be made for glycogens and starches from other sources. We will attempt in muscle to detect and characterize the enzyme(s) that synthesize the protein to carbohydrate linkage. We will determine the nature of the phosphodiester and the location of the phospho-esters in the glycogen molecule as well as exploring the enzymic mechanisms whereby they are incorporated into glycogen. We will prepare and use antibodies to locate glycogenin muscle and other sources of glycogen. We will use the immature frog oocyte for in vivo studies of the synthesis of glycogenin and glycogen as well as the incorporation of phosphate esters.
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