Over the previous grant period, the rod outer segment (ROS) plasma membrane has been isolated from disk membranes and shown to contain a number of major proteins not present in disk membranes. These include the cGMP-gated cation channel, Na+Ca++ exchange protein, a 240kD spectrin-like protein, and a 38 kD membrane associated protein. In this proposal the molecular properties of these and other plasma membrane proteins will be studied in detail in order to elucidate their role in visual transduction process and other important metabolic and structural functions in rod and cone outer segments. Monoclonal antibodies (Mabs) recently produced in this laboratory will be used with biochemical, immunochemical, microscopic, molecular biology and reconstitution techniques to define the structural, functional and antigenic domains of these proteins and to compare their properties with corresponding proteins in cone photoreceptor cells and other cell types. Proposed areas of research are as follows: (1) The antigenic binding sites for anti cGMP-gated channel Mabs (PMcIDI,PMc 2GII, others) will be determined by recombinant DNA techniques, radioimmune assays (RIA) using synthetic peptides and immunocytochemical methods to determine the topography of the 63kD channel in ROS plasma membranes. Site-directed antibodies against known sequences will also be prepared and their binding cites localized by immunogold-labeling studies. The effect of these antibodies on the cGMP-gated channel activity will be studies reconstituted vesicle systems. (2) The 230KD NaCa exchange protein will be studied using a number of anti-230KD monoclonal antibodies we have recently generated. (3) A 240KD spectrin-like protein associated with the cGMP gated channel will be partially sequenced for comparison to other spectrin cytoskeletal proteins and the morphological features of the isolated 240 KD protein will be studied by electron microscopy. (4) A major plasma membrane associated 38 KD protein will be isolated and its molecular properties and its interaction with other plasma membrane proteins will be studied. (5) Rhodopsin from plasma membrane and disk membranes will be purified by immunoaffinity chromatography. The C-terminal, N-terminal segments, and tryptic peptide maps of these rhodopsins will be compared in order to determine if plasma membrane and disk membrane rhodopsin are identical or if they undergo differential post-translational modification as part of the sorting process.
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