Abstract
Abstract: :
Purpose: The association of α–crystallin to lens membranes increases with age and cataract. Lipid compositional changes also occur with age, cataract, and diabetes. In this study we determined the influence of lipid compositional differences on the binding capacity of α–crystallin to lipid vesicles in–vitro. Methods: Lipids were extracted from pools of younger (22 y, n=30) and older human (69 y, n=26) lenses. In addition, pools of lenses from non–insulin–taking diabetics (58 y, n=20) and from diabetics who had taken insulin for an average of 6 years (59 y, n=26) were also analyzed. Extracted lipids were extruded into large unilamellar vesicles. α–Crystallin was mixed with the lipid at 36oC, allowed to bind for about 12 hours, and centrifuged at 14,000 g. This centrifugal force was low enough not to pellet free α–crystallin, but high enough to pellet the lipid. α–Crystallin–lipid binding was characterized by comparing the amount α–crystallin in the pellets of samples with and without lipid. Protein was measured using an assay that minimized interference from lipids. Results: The binding capacity of α–crystallin to lipids was 12, 19, 8.9, 17 µg bound/mg lipid for lens lipids extracted from the following respective groups: young, old, diabetics on insulin, and diabetics not on insulin. The amount of α–crystallin in the pellet (bound α–crystallin) was significantly higher for the lipids from the older pool of lenses compared with the younger pool, p = 0.37. α–Crystallin binding capacity was also significantly higher in non–insulin–taking diabetics compared with the insulin–taking diabetic group (p=0.011). Conclusions: The binding capacity of α–crystallin to lens lipids increases with age and is increased in lens lipids from non–insulin–taking diabetics compared to lens lipids from insulin–taking diabetics. Our data support the hypothesis that with diabetes or age, more α–crystallin binds to the membrane thus creating condensation points for further crystallin binding and protein oxidation. Because the lens membrane contains much more oxygen and sensitizers than the surrounding cytoplasm, the susceptibility of membrane–bound proteins to oxidation is enhanced. EYO7975 (DB), and an unrestricted grant from Research to Prevent Blindness Inc., NY.
Keywords: lipids • cataract • oxidation/oxidative or free radical damage