The overall objective of the proposed project is to cure ocular diabetic diseases, diabetic retinopathy and diabetic cataract, and corneal re-epithelialisation following vitrectomy in retinopathy. We need to know how the sorbitol pathway functions in diabetes, and how effectors of the enzymes of the sorbitol pathway, aldose reductase and polyol dehydrogenase alter the catalytic mechanism of these enzymes to alleviate these ocular diseases. We will use our methods to isolate these enzymes by chromatography from rat, bovine and human lenses and corneas, and human lenses, corneas, placentas and erythrocytes. A detailed study of the kinetics and mechanism of aldose reductase and polyol dehydrogenase will be carried out using the reaction rate spectrophotometer requested, by the research assistant requested. This will lead to a description of the catalytic mechanism and kinetic rate constants for these enzymes. The kinetics of aldose reductase inhibition will be studied. This will show the molecular basis of action of these inhibitors for each species, and lead to the design of better effectors of the polyol pathway by comparing the results obtained in this study with those from pharmacological tests on experimental animals and clinical trials. Kinetic parameters will be studied over the widest possible ranges of substrate concentration, pH, temperature, ionic strength, dielectric constant and viscosity, using variety of physiological substrates, The results will be assessed by computer curvefitting procedures that I have developed for this project. Studies on the chemical nature of catalysis by aldose reductase will be carried out by trapping Schniff-base intermediates formed between ligands and enzymes by reduction with borohydride (tritiated) and measuring radioactive incorporation. Further experiments, using enzyme digestion, chromatography of the products and amino acid analysis would show the nature of the intermediate and its location on the enzyme surface.