AGE-RELATED-CHANGES OF GLYCOSIDASES IN HUMAN RETINAL-PIGMENT EPITHELIUM

This study was undertaken to determine whether there are age-related c hanges in the specific activities of several glycosidases in fresh ret inal pigment epithelial cells (RPE) isolated from the posterior pole o f human donor eyes. One hundred and twenty-one pairs of eyes from huma n donors, between the ages of 43 and 95 years, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and the Cleveland Ohio Eye Bank within 18 to 24 h of death. None had historie s of diabetes, hepatitis, HIV infection, intraocular surgery, or docum ented age-related macular degeneration, although several older donors with evidence of drusen were included in the study. RPE cells were iso lated from the posterior third of the retina using the conventional ru sh method and homogenized with a glass, Broeck tissue grinder. All pos t-nuclear supernatants were anlayzed for glycosidase activity; a small er number of nuclear pellets were assayed to verify that the majority of the enzyme activity was associated with the post-nuclear sypernatan ts. Glycosidase activity was quantitated fluorometrically by measuring the enzymatic release of umbelliferone from synthetic substrate prepa rations, specific for each enzyme. Total protein was determined by a m icro BCA protein assay. Regression analysis revealed statistically sig nificant age-related decreases for the specific activities of alpha-ma nnosidase (p = 0.0001), beta-galactosidase (p = 0.0001), N-acetyl-beta -glucosaminidase (p = 0.0001), and N-acetyl beta galactosaminidase (p = 0.0001) in fresh human donor RPE cells taken from the region of the posterior third of the retina that included the macula. Mannose and N- acetyl-glucosamine are major carbohydrate monomers of the oligosaccari de chains of human rhodopsin, and a realtively high percentage of the oligosaccharide chains are galactosylated. Defects in their degradatio n may lead to the accumulation of undigested residual material in the RPE.