Grami Vahid, Marrero Yernan, Huang Li, Tang Daxin, Yappert Marta C, Borchman Douglas
Department of Ophthalmology and Visual Science, Kentucky Lions Eye Center, University of Louisville, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202, USA.
Exp Eye Res. 2005 Aug;81(2):138-46. doi: 10.1016/j.exer.2004.12.014.
The association of alpha-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 alpha-crystallin to lipid vesicles in vitro. Lipids were extracted from pools of human lenses from younger (22+/-4 y, n=30) and older (69+/-3 y, n=26) nondiabetic donors as well as from diabetics taking insulin (60+/-9 y, n=26) and diabetics not taking insulin (58+/-9 y, n=20). Diabetics were insulin dependent for an average of 6 years. Extracted lipids were extruded into large unilamellar vesicles. alpha-Crystallin was mixed with the lipid at 36 degrees C, allowed to bind for about 12 h, and centrifuged at 14,000 g. This centrifugal force was low enough to not pellet free alpha-crystallin but high enough to pellet the lipid and bound alpha-crystallin. alpha-Crystallin-lipid binding was characterized by comparing the amount alpha-crystallin in the pellets of samples with and without lipid. Protein was measured using an assay that minimized interference from lipids. Lipid composition was determined by 31P-NMR spectroscopy. The binding capacity of alpha-crystallin to lipids was 12, 19, 8.9, 17 microg bound/mg lipid for lens lipids extracted from younger, older, insulin-treated and nontreated diabetic donors, respectively. The amount of alpha-crystallin in the pellet (bound alpha-crystallin) was significantly lower for the lipids from the younger group of lenses, p=0.033 and insulin-treated group, p=0.006, compared with the older group of lenses. Higher binding capacity was associated with a higher relative amount of sphingolipid and lower relative amounts of phosphatidylethanolamine-related lipid and phosphatidylcholine. The binding capacity of alpha-crystallin to lens lipids, measured in vitro, increases with age and decreases in diabetic donors that were treated with insulin. Our data support the idea that with age and perhaps certain types of diabetes, more alpha-crystallin is bound to the membrane and serves as a condensation point to which other crystallins bind and then become oxidized.
α-晶体蛋白与晶状体膜的结合随年龄增长和白内障的发生而增加。脂质组成也会随着年龄、白内障和糖尿病而发生变化。在本研究中，我们测定了脂质组成差异对α-晶体蛋白在体外与脂质囊泡结合能力的影响。脂质从年轻（22±4岁，n = 30）和年老（69±3岁，n = 26）非糖尿病供体的人晶状体库中提取，以及从接受胰岛素治疗的糖尿病患者（60±9岁，n = 26）和未接受胰岛素治疗的糖尿病患者（58±9岁，n = 20）中提取。糖尿病患者平均依赖胰岛素6年。提取的脂质被挤压成大单层囊泡。α-晶体蛋白在36℃与脂质混合，使其结合约12小时，然后在14,000 g下离心。这种离心力足够低，不会使游离的α-晶体蛋白沉淀，但又足够高，能使脂质和结合的α-晶体蛋白沉淀。通过比较有脂质和无脂质样品沉淀中α-晶体蛋白的量来表征α-晶体蛋白与脂质的结合。使用一种能使脂质干扰最小化的检测方法来测量蛋白质。通过31P-NMR光谱法测定脂质组成。从年轻、年老、接受胰岛素治疗和未接受胰岛素治疗的糖尿病供体提取的晶状体脂质中，α-晶体蛋白与脂质的结合能力分别为12、19、8.9、17 μg结合/mg脂质。与年老组晶状体相比，年轻组晶状体脂质沉淀中（结合的α-晶体蛋白）α-晶体蛋白的量显著降低，p = 0.033；胰岛素治疗组，p = 0.006。较高的结合能力与较高的鞘脂相对含量以及较低的磷脂酰乙醇胺相关脂质和磷脂酰胆碱相对含量有关。体外测定的α-晶体蛋白与晶状体脂质的结合能力随年龄增长而增加，在接受胰岛素治疗的糖尿病供体中降低。我们的数据支持这样一种观点，即随着年龄增长以及可能某些类型的糖尿病，更多的α-晶体蛋白与膜结合，并作为其他晶体蛋白结合然后被氧化的凝聚点。
