Lieska N, Yang H Y, Maisel H
Department of Anatomy, University of Illinois College of Medicine, Chicago 60612.
Curr Eye Res. 1991 Nov;10(11):1037-48. doi: 10.3109/02713689109020342.
The beaded-chain filaments unique to the fiber cells of the crystalline lens are composed of a linear array of spheroidal particles which appear to be connected by a filamentous backbone. In order to determine the existence of the putative backbone and to characterize its constituents, one of the major proteins associated with beaded-chains in the chicken lens was investigated. 49kD was isolated in an enriched fraction derived from the 8M urea extract of the lens cell water-insoluble residue. The polypeptide (which exists in several charge isoforms, the major at pI 5.2) was purified sequentially by gel filtration on Sephacryl S-200, hydrophobic interaction chromatography on phenyl-Sepharose, and anionic exchange chromatography on Mono Q, all under denaturing conditions. Immunoblot analyses established that 49kD was immunologically distinct from vimentin, actin, and tubulin/MAPs (representing the three classes of cytoplasmic filaments), as well as from the crystallins. Amino acid analyses demonstrated compositional differences for 49kD compared with lens actin and vimentin, and one- and two-dimensional peptide mapping of 49kD and vimentin revealed no homology. Electron microscopy demonstrated that short, contorted filaments were produced upon removal of purified 49kD from urea to low-salt buffers. In the presence of physiological salt concentrations 49kD assembled into extensive 4-6nm diameter, straight filaments similar to the backbone seen in native beaded-chain filaments, but morphologically distinct from the other cytoplasmic filament classes.
晶状体纤维细胞特有的串珠链细丝由一系列呈线性排列的球状颗粒组成，这些颗粒似乎由丝状主链连接。为了确定假定主链的存在并表征其成分，对鸡晶状体中与串珠链相关的一种主要蛋白质进行了研究。在从晶状体细胞水不溶性残渣的8M尿素提取物中获得的富集级分中分离出了49kD。该多肽（存在几种电荷异构体，主要异构体的pI为5.2）在变性条件下，依次通过Sephacryl S - 200凝胶过滤、苯基琼脂糖疏水相互作用色谱和Mono Q阴离子交换色谱进行纯化。免疫印迹分析表明，49kD在免疫上与波形蛋白、肌动蛋白和微管蛋白/MAPs（代表三类细胞质细丝）以及晶状体蛋白不同。氨基酸分析表明，与晶状体肌动蛋白和波形蛋白相比，49kD的组成存在差异，49kD和波形蛋白的一维和二维肽图谱显示没有同源性。电子显微镜显示，将纯化的49kD从尿素转移到低盐缓冲液后会产生短而扭曲的细丝。在生理盐浓度下，49kD组装成直径为4 - 6nm的广泛的直细丝，类似于天然串珠链细丝中看到的主链，但在形态上与其他细胞质细丝类别不同。
