The long term objective of this research program is to study the molecular structure and properties of rod outer segment (ROS) disk and plasma membrane proteins in order to elucidate their role in visual excitation, in the organization and renewal of the ROS organelle and in retinal degenerative diseases. Four related areas of research will be investigated in this grant period: 1) The ROS plasma membrane and plasma membrane specific proteins will be purified and characterized. This will be carried out by labeling ROS plasma membrane specific glycoproteins with recently developed Ricin-gold-dextran particles and separating the 'dense' labeled plasma membrane from disks by gradient centrifugation. The protein and lipid composition of the plasma membrane will be determined and compared with disk membranes using biochemical, immunochemical and electron microscopic techniques developed over the past grant period. Emphasis will be directed toward identifying and characterizing the c-GMP binding protein, Na+ channels and ROS plasma membrane-specific glycoproteins. The generation of monoclonal antibodies (Mab) against these proteins for use as specific probes for studying their distribution, structure and function will be carried out. 2) ROS spectrin recently identified in this laboratory will be purified by gel filtration, ion exchange and immunoaffinity chromatography and characterized with regard to its subunit structure by SDS-gel electrophoresis, its size and shape by Platinum rotary shadowing for electron microscopy and its interaction with other membrane and cytoskeletal proteins by binding analysis and reconstitution methods. The possible role of ROS spectrin and other cytoskeletal proteins in disk-disk and disk-plasma membrane interactions will also be studied. (3)Peripherin, a newly discovered ROS rim protein, will be further characterized using biochemical and DNA recombinant techniques. The gene will be cloned and its nucleotide and amino acid sequence determined in order to obtain information about its membrane organization and relation to other known proteins. 4) Antirhodopsin Mabs will be further characterized and used as immunological probes to analyze the structural and functional domains of rhodopsin.
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