Ubiquitin (Ub), a protein of 8.5 kD, is encountered in all eukaryotic cells. It is involved in covalent protein modifications leading either to proteolytic degradation by a specialized, ATP- dependent system or to accumulation of conjugates, the function of which is poorly understood. The process of conjugation and degradation via the Ub pathway has been described only in a limited number of biological systems. A novel study of the functions of Ub in rat retina is proposed based on the observation of Ub-dependent proteolysis and conjugation to rat retina proteins. The long-term objectives involve localization and quantitation of the activities in retina cells, identifying the cell types and target proteins for Ub-dependent proteolysis and/or conjugation under normal (light/dark cycle, young and old, pigmented and albino animals) and pathological conditions (light damage, inborn retinal dystrophy) of rat retina. In this pilot study, the Ub-dependent proteolysis of a specific exogenous substrate, radioiodinated bovine serum albumin, will be measured by quantitating TCA-soluble counts to find a retinal fraction (e.g., soluble, membrane, rod outer segments) of highest activity. In that fraction, Ub-dependent degradation of endogenous proteins will be assayed by gel electrophoresis and densitometric scanning. The proteins undergoing significant degradation will be electroeluted from preparative gels for identification. The conjugate formation between retinal proteins and Ub will be studied by Western blotting of the proteins resolved by gel electrophoresis followed by detection with antibody against Ub conjugates. To find out how a particular state (normal, pathological) of the retina affects the most dominant conjugate formation, the conjugated protein(s) will be electroeluted from the gel, treated with isopeptidase to remove the Ub moiety, radiolabeled and added to fractionated retinas obtained from normal and defective animals subjected to dark/light treatment. The conjugate formation will be detected by autoradiography. The study will allow us to better understand the role of Ub in protein modification and turnover in rat retina. Further steps will be undertaken to establish the relationship between Ub-dependent processes and the known physiological responses of the retina and its individual cell types.
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