Ketis N V, Hoover R L, Karnovsky M J
J Cell Physiol. 1986 Aug;128(2):162-70. doi: 10.1002/jcp.1041280205.
The plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane-associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane-associated proteins and extraction with 1% Triton X-100, 60%. At least four heavily glycosylated bands (185-, 165-, 150-, and 130,000 mol-wt) were evident. The Triton-insoluble pellet fraction contained several major polypeptides (30-, 43-, 58-, and 240,000 mol-wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol-st) and vimentin (58,000 mol-wt). Strikingly, vimentin and a 240,000 mol-wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1-like and a 4.1-like protein associated with plasma membranes. The 2.1-like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1-like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti-2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti-protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern. This paper presents several novel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: the presence of two erythroid-like cytoskeletal polypeptides; the presence of vimentin and a 240,000 mol-wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co-elution in a 4:1 mol ratio with a protein perturbant; and the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.
牛主动脉内皮细胞质膜被分离、鉴定，并发现其含有至少四种与膜相关的细胞骨架蛋白。将质膜暴露于盐介质（高达1M KCl）中，导致30%的与质膜相关的总蛋白释放，用1% Triton X - 100提取时，释放量为60%。至少有四条高度糖基化的条带（分子量分别为185000、165000、150000和130000）很明显。Triton不溶性沉淀部分含有几种主要多肽（分子量分别为30000、43000、58000和240000），其中两种通过免疫印迹鉴定为细胞质肌动蛋白（43000分子量）和波形蛋白（58000分子量）。引人注目的是，在超过60%的质膜制剂中，波形蛋白和一种240000分子量的多肽通常以大约4:1的摩尔比存在。我们还报告了与质膜相关的一种2.1样蛋白和一种4.1样蛋白的存在。2.1样蛋白表现出与红细胞蛋白2.1相似的溶解度和表观分子量（210000）。同样，内皮4.1样蛋白表现出与红细胞蛋白4.1相似的溶解度和表观分子量。用抗2.1抗体对固定和通透的培养物进行免疫荧光染色显示出纤维状模式。相比之下，用抗4.1蛋白染色的细胞发出明亮荧光，呈现弥漫性和点状模式。本文提出了一些关于牛主动脉内皮细胞质膜组成的新观察结果，即：存在两种类红细胞细胞骨架多肽；在超过60%的质膜制剂中，波形蛋白和一种240000分子量的多肽以4:1的摩尔比存在，并与一种蛋白质干扰剂以4:1的摩尔比共洗脱；以及无法从质膜制剂中释放肌动蛋白，这表明肌动蛋白与质膜制剂中的其他分子相关联。
