The biosynthesis of retinal cell membranes is being explored using a newly developed technique of immunochemical subcellular fractionation termed immunoaffinity partitioning. Biotin conjugated antibody to frog or bovine opsin will be reacted with retinal homogenates to bind to all components bearing newly synthesized opsin. Separation of the antibody-coated particles will be achieved by partitioning in an aqueous polymer two-phase system composed of dextran and polyethylene oxide. Specific partitioning to the top (polyethylene oxide rich) phase will be investigated by an affinity probe consisting of streptavidin (a bacterial biotin binding protein) and biotinyl-polyethylene oxide. Radioactive amino acid precursors will be incorporated for short periods (30-90 min) to label opsin as it resides in the rough endoplasmic reticulum, Golgi or in the post-Golgi smooth membranes during its vectorial transport to the rod outer segment. Specific antibodies to unique domains of opsin are being developed by the monoclonal hybridoma technique. Their specificity will be evaluated by two-dimensional immunoelectrophoresis to be used as probes of the orientation of opsin during its transport. Specific peptides of opsin will be exploited as inhibitors to identify the specificity of the monoclonal antibodies. Colonies of mice (rds) and rats (Wag/Rij) will be expanded to distribute breeding pairs to interested investigators. These rats will be investigated for disorders of membrane protein biosynthesis
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