Our long term goal is to elucidate the biochemical and biophysical mechanisms underlying visual excitation and photoreceptor metabolism. Our approach is to attack important, related problems at several organizational levels of the photoreceptor cell. These levels are first, the visual pigments themselves, especially chicken cone pigments where, besides establishing their amino acid sequences, we plan to purify and study the pigments biophysically (vibrational spectra, bleaching kinetics). Second, at the level of the action of light on the pigments, we will seek to learn the nature of the storage of the photon's energy by the primary photoproduct, bathorhodopsin, and the properties of metarhodopsin II which allows it to interact with and activate the GTP binding protein, transducin. Third is the level of the photoreceptor membrane. We want to measure and study the control of the surface potential of photoreceptor membranes, since the surface potential will have profound effects on the concentration of charged substrates (e.g. cGMP), co-factors (e.g. GRP), and enzymes (e.g. transducin) near the surface of photoreceptor membranes where activation and some subsequent enzymatic events takes place. We also plan on clarifying the topology of rhodopsin in the membrane and will purify cone photoreceptor membranes. Fourth, is the level photoreceptor cell, especially with regard to the transduction machinery itself, and the transmitters and other key small molecules within the retinal cells. Here we will utilize new approaches to transducin (Raman difference spectroscopy, cation binding, meta II initiated conformational changes) and the use of phosphorus 31 NMR spectroscopy to study intracellular transmitters and metabolites in vivo. This integrated approach will offer not only new insight into visual excitation and the control of retinal metabolism, but also lead to the development of new and more sensitive ways to assay the health and metabolic state of the retina in vivo.
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