The goal of the proposed research is to elucidate the molecular mechanism of visual excitation in vertebrate retinal rod cells. Phototransduction is thought to involve a light-triggered enzyme cascade that leads to a rapid reduction of cGMP level in the rod outer segments (ROS). Central to this process is transducin (GTPase), a signal-carrier protein responsible for coupling the phosphodiesterase to rhodopsin. It is evident that in order to understand the molecular basis of the cascade, detailed information concerning the structure, dynamics, and function of the transducin is needed.
The specific aim of this study is to gain an understanding of the molecular basis of transducin activation through detailed structural, biochemical and immunological analysis. We proposed to carry out the following studies: (1) Chemical modification, proteolysis, and photoaffinity labeling will be carried out to analyze the structure of the transducin subunits and the amino acid residues of the GTP binding site. (2) Fluorescence spectroscopy will be used to measure the conformational change of transducin, the rate of GTP-GDP exchange, the molecular dimensions of transducin, and the prosimity relationship between rhodopsin and transducin. (3) Monospecific anti-transducin antibodies will be used to identify """"""""transducin-like"""""""" proteins in retinas of different species and to purified transducin mRNA. The use of antibodies to identify and isolate the functional sites of transducin will be explored. (4) The interaction of transducin with phosphodiesterase, rhodopsin, and the ROS lipids will be systematically investigated. (5) Finally, an attempt will be made to isolate the plasma membranes of ROS by a novel method involving the use of impermeable fluorescent probes, monoclonal antibodies and affinity chromatography. The chemical composition and ionic properties of the purified plasma membranes will be characterized. Preliminary experiments will be carried out to define the actions of cGMP and Ca ions on the permeability of the plasma membranes. By advancing our knowledge in these five specific areas, our research is expected to contribute to the overal goal of achieving a better understanding of the molecular basis of visual transduction.
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