The regulation of the fluxes and metabolism of vitamin A in the retina and retinal pigment epithelium is not yet well understood and is the subject of this grant request. Central to this problem are quantitative studies on certain enzymes and binding proteins which process vitamin A in the eye and which could be points of regulation. In this grant request we propose to study by chemical and biochemical means retinal ester esterase, synthetase, rhodopsin and certain of the retinol(al) binding proteins. The two enzymes will be purified to homogeniety and their mechanisms of action determined. In order to study these mechanisms a new quantitative assay system for these enzymes will be developed involving the transfer of the retinoids from small unilamellar vesicles (SUV's) to the protein. This assay will allow for the precise calculation of retinoid concentrations as well as providing a known and stable environment for them which allows for a meaningful calculation of thermodynamic and kinetic constants. This assay system should be generally useful for binding studies involving hydrophobic ligands. Having a quantitative assay for the enzymes in hand, we will determine whether they are regulated by light in the albino rat and frog. Highly specific, irreversible, mechanism-based inhibitors will be synthesized for these enzymes and they will be tested in vivo to determine their physiological roles. The same assay system mentioned above will be used to quantitatively measure the binding of the retinol(al)s to ascertain the retinol binding proteins and determine whether the activity of these proteins is regulated by light. Specific irreversible inactivators of these proteins will also be prepared and studied in vivo. Finally, new specific mechanism-based inactivators for opsin have been prepared. These will be primarily used to study how rhodopsins activity is regulated by light. Quantitative studies on the visible absorption spectra of these inhibitor(s) - opsin complexes will also be of interest in determining the mechanism(s) by which opsin perturbs the absorption spectra of bound chromophores.
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