Yemelyanov A Y, Katz N B, Bernstein P S
Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
Exp Eye Res. 2001 Apr;72(4):381-92. doi: 10.1006/exer.2000.0965.
The macula of the human retina contains extraordinarily high concentrations of lutein and zeaxanthin, xanthophyll carotenoids that appear to play an important role in protecting against age-related macular degeneration, the leading cause of blindness among the elderly. It is likely that the uptake and stabilization of these carotenoids is mediated by specific xanthophyll-binding proteins. In order to purify and characterize such a binding protein, a carotenoid-rich membrane fraction derived from human macula or peripheral retina was prepared by homogenization, differential centrifugation, and detergent solubilization. Further purification was carried out using ion-exchange chromatography and gel-filtration chromatography coupled with continuous photodiode-array monitoring for endogenously associated xanthophyll carotenoids. The most highly purified preparations contained two major protein bands at 25 and 55 kDa that consistently co-eluted with endogenous lutein and zeaxanthin. The visible absorbance spectrum of the binding protein preparation closely matches the spectral absorbance of the human macular pigment, and it is bathochromically shifted about 10 nm from the spectrum of lutein and zeaxanthin dissolved in organic solvents. Binding of exogenously added lutein and zeaxanthin is saturable and specific with an apparent Kd of approximately 1 microM. Canthaxanthin and beta-carotene exhibit no significant binding activity to solubilized retinal membrane proteins when assayed under identical conditions. Other potential mammalian xanthophyll-binding proteins such as albumin, tubulin, lactoglobulin and serum lipoproteins possess only weak non-specific binding affinity for carotenoids when assayed under the same stringent binding conditions. This investigation provides the first direct evidence for the existence of specific xanthophyll-binding protein(s) in the vertebrate retina and macula. The possible roles of xanthophyll-binding proteins in normal macular function and in the pathogenesis of age-related macular degeneration remain to be elucidated.
人类视网膜的黄斑区含有极高浓度的叶黄素和玉米黄质，这两种叶黄素类胡萝卜素似乎在预防年龄相关性黄斑变性方面发挥着重要作用，而年龄相关性黄斑变性是老年人失明的主要原因。这些类胡萝卜素的摄取和稳定可能是由特定的叶黄素结合蛋白介导的。为了纯化和鉴定这种结合蛋白，通过匀浆、差速离心和去污剂增溶制备了来自人类黄斑或周边视网膜的富含类胡萝卜素的膜组分。使用离子交换色谱和凝胶过滤色谱，并结合连续光电二极管阵列监测内源性相关的叶黄素类胡萝卜素进行进一步纯化。纯化程度最高的制剂含有两条主要蛋白带，分子量分别为25 kDa和55 kDa，它们始终与内源性叶黄素和玉米黄质共洗脱。结合蛋白制剂的可见吸收光谱与人类黄斑色素的光谱吸收密切匹配，并且相对于溶解在有机溶剂中的叶黄素和玉米黄质的光谱，其红移约10 nm。外源添加的叶黄素和玉米黄质的结合是饱和且特异的，表观解离常数约为1 μM。在相同条件下测定时，角黄素和β-胡萝卜素对溶解的视网膜膜蛋白没有明显的结合活性。在相同严格的结合条件下测定时，其他潜在的哺乳动物叶黄素结合蛋白，如白蛋白、微管蛋白、乳球蛋白和血清脂蛋白，对类胡萝卜素仅具有弱的非特异性结合亲和力。这项研究为脊椎动物视网膜和黄斑中存在特异性叶黄素结合蛋白提供了首个直接证据。叶黄素结合蛋白在正常黄斑功能和年龄相关性黄斑变性发病机制中的可能作用仍有待阐明。
