Characterization of α-crystallin-plasma membrane binding
Characterization of α-crystallin-plasma membrane binding
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DOI：
10.1074/jbc.275.9.6664
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发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Petrash, JM
中科院分区：
文献类型：
--
作者：
Cobb, BA;Petrash, JM
Alpha-Crystallin是一种由两个相关亚单位(Alpha A-和Alpha B-Crystallin)组成的大型晶状体蛋白复合体，已知随着年龄和/或白内障的发生，它与纤维细胞质膜的结合越来越紧密，为了更好地了解结合机制，我们建立了一种灵敏的膜结合分析方法，使用晶状体质膜和连接到小荧光标记(Alexa350(R))的重组人αA和Alpha B晶体蛋白。αA和αB均聚络合物以及重组的3：1杂多络合物以特定的、可饱和的和部分不可逆的方式结合到晶状体膜上，这种方式对时间和温度都很敏感。在酸性pH条件下和去除暴露的固有膜蛋白结构域后，与膜结合的α-晶体蛋白的量增加，但在高离子强度下不受影响，表明α-晶体蛋白主要通过疏水相互作用与纤维细胞质膜结合。测得重组的3：1异构体复合体的结合容量和亲和力分别为3.45+/-0.11 ng/µg膜和4.57+/-0.50×10(-4)µg(-1)膜，膜结合数据支持混合α-晶状体蛋白复合体的物理性质可能与晶状体内α-晶状体蛋白的功能特别相关的假设。
alpha-Crystallin, a large lenticular protein complex made up of two related subunits (alpha A- and alpha B-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract, To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human alpha A- and alpha B-crystallins conjugated to a small fluorescent tag (Alexa350(R)). Both alpha A and alpha B homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of alpha-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that alpha-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3.45 +/- 0.11 ng/mu g of membrane and 4.57 +/- 0.50 x 10(-4) mu g(-1) of membrane, respectively, The present membrane binding data support the hypothesis that the physical properties of a mixed alpha-crystallin complex may hold particular relevance for the function of alpha-crystallin within the lens.