The overall goal of my research is to understand the structure and function of the junctional sarcoplasmic reticulum membrane of skeletal and cardiac muscle. My approach to the study of this specialized region of the sarcoplasmic reticulum is through purification and characterization of the protein constituents of this membrane. In skeletal muscle, the sarcoplasmic reticulum consists of three major proteins: Ca++ + Mg++ ATPase, calsequestrin and Mr 53,000 glycoprotein. Indirect immunofluorescence has shown us that both calsequestrin and Mr 53,000 glycoprotein are localized in the region of the junctional sarcoplasmic reticulum membrane. The proposed research concerns the structure, function and morphology of the Mr 53,000 glycoprotein. Lectin affinity chromatography will be used to purify the Mr 53,000 glycoprotein from rabbit skeletal muscle sarcoplasmic reticulum. Limited proteolysis will be performed to fragment the glycoprotein into two or three fragments which will then be purified using SDS chromatrography. Amino acid, carbohydrate, N-terminal sequence and COOH-terminal sequence analysis will be performed on purified fragments and the proteolytic fragments will be aligned NH2-terminal to COOH-terminal. Location of the sites of carbohydrate attachment, the site(s) of 8-N3-ATP labeling and the cytoplasmic associated domains in the proteolytic fragments of Mr 53,000 glycoprotein will be defined. Characterization of 8-N3-ATP binding will be completed and the functional role of the ATP binding site on the Mr 53,000 glycoprotein will be investigated. Ca++ binding properties of the Mr 53,000 glycoprotein will also be determined. The Mr 160,000 glycoprotein of the sarcoplasmic reticulum will be purified and compared to the Mr 53,000 glycoprotein. Experiments will be performed to test the hypothesis that the Mr 160,000 glycoprotein is a product of an inter-polypeptide cross-link between the Mr 53,000 glycoprotein and a sarcoplasmic reticulum or transverse tubular protein. Indirect immunoferritin labeling of ultrathin frozen sections will determine the ultrastructural localization of the Mr 53,000 glycoprotein in adult rat skeleta and papillary muscle. Finally, a preliminary characterization of the Mr 53,000 glycoprotein from canine cardiac sarocplasmic reticulum will be performed.
