THE PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PROTEINS OF THE SQUID RETINA
Abstract
In order to learn about the molecular events involved in visual excitation, a set of monoclonal antibodies was developed against photoreceptor-specific proteins in the squid (Lolliguncula brevis). The visual pigment of the squid, rhodopsin, was of particular interest. Rhabdomeric membranes were analyzed by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate. Opsin is resolved as a diffuse band at approximately 47 kilodaltons (kd), with oligomers of opsin at higher molecular weights. Further analysis reveals a second polypeptide present within the diffuse band at 47 kd. The second polypeptide: (1) runs as a sharp band at approximately 45 kd, (2) is a membrane protein specific to the photoreceptor, (3) emits green fluorescence when rhabdomeric membrane extracts are reduced with sodium borohydride prior to electrophoresis, and (4) does not possess a binding site for Concanavalin A. Monoclonal antibodies were made by the hybrid myeloma technique, using squid retina fragments as the immunogen. Initial screening was by enzyme linked immunosorbent assay against such fragments. Further definition of the specificity of the antibodies was made by observing the binding pattern to retina proteins resolved by electrophoresis and transferred to nitrocellulose sheets. Monoclonal antibodies were found which bind specifically to opsin and its oligomers, and to the 45 kd polypeptide. The cellular localization of these two antigens was determined by indirect immunocytochemistry on frozen retina sections. Monoclonal antibodies specific for opsin were localized to the outer segments of the photoreceptors, while antibodies specific for the 45 kd polypeptide were localized throughout the entire photoreceptor. These results suggest that the second polypeptide is a form of opsin. In this case, it differs from the usual form in its Concanavalin A binding properties and distribution in the photoreceptor. The usual form of opsin, localized to the outer segment, is evidently involved in phototransduction. Possibly, the 45 kd polypeptide is a form of opsin involved in the synthesis or degradation of the usual form, and may serve no role in phototransduction.
Degree
Ph.D.
Subject Area
Biology|Aquaculture|Fish production
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