Molecular properties and structural organization of proteins in retinal rod outer segment (ROS) disk and plasma membranes will be studied to elucidate their functional role in visual excitation and cell-cell recognition and regulation in normal and retinal degenerative disease states. We will study the molecular properties of the high Mr proteins of disk membranes and define their possible role as structural and functional elements in disk-disk and disk-plasma membrane interactions. Monoclonal antibodies will be used with solid-phase radioimmune assays, immunoaffinity chromatography, immunospecific cell membrane labeling and biochemical analysis; i.e. electrophoresis, HPLC peptide mapping, amino acid analysis etc. to purify, quantify and characterize ROS membrane proteins. Their molecular properties and interaction with other proteins including actin and calmodulin will be studied and correlated with the molecular properties of potentially-related cytoskeletal proteins (myosin and spectrin) of other cell types. The effect of limited proteolytic digestion, divalent cations, ionic strength and addition of specific ROS cytoplasmic proteins on the association-dissociation behavior of isolated disks will be studied and correlated with changes in the properties of high Mr proteins and other proteins of disk membranes. Monoclonal antibodies against specific regions of rhodopsin will be characterized with the aim of using these reagents to probe structural organization, conformational changes and functional domains of rhodopsin in disk membranes. Identification and characterization of antigenic sites on rhodopsin will be determined by limited proteolytic digestion of rhodopsin and synthetic peptides in conjunction with RIA assays, HPLC separations, amino acid analysis and protein binding and ion transport assays. ROS plasma membrane will be isolated for analysis of its structural and functional properties. Lectins and monoclonal antibodies will be used in conjunction with newly developed ferromagnetic iron dextran and gold-dextran microspheres to specifically label the outer surface of ROS plasma membranes and separate these membranes from disk membranes by affinity magnetic chromatography and/or affinity density perturbation. Purify and sideness of the ROS plasma membranes will be studied by RIA and lectin binding assays and factors such as Ca++, cGMP, ROS cytoplasmic proteins, etc. affecting Na efflux from these vesicles will be studied. Protein components of Ros plama membranes will also be studied by immunochemical, biochemical and microscopic techniques and compared with those present in disk membranes.
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