Properties of smooth muscle meta-vinculin.
Properties of smooth muscle meta-vinculin.
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DOI：
10.1083/jcb.104.3.473
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发表时间：
1987-03
影响因子：
7.8
通讯作者：
Craig, S W
中科院分区：
文献类型：
--
作者：
Siliciano, J D;Craig, S W
定量研究表明，偏纽蛋白在0.6M盐中的溶解度是用于提取纽蛋白的0.01M盐缓冲液中的九倍。基于这一发现，我们开发了一种从肌肌高盐提取液中以43%的得率和98%的纯度纯化偏纽蛋白的方法。与我们早先在不固定的冰冻切片(30)上进行的提取研究不同，目前在组织匀浆上进行的研究表明，偏纽素的增溶不需要非离子洗涤剂。此外，纯化或部分纯化的偏纽蛋白都不能与Triton X-114胶束结合。纯化的偏纽蛋白是一种不对称的单体分子，斯托克斯半径为50.9A，沉降系数为6.35S，摩擦比为1.46。计算得到的偏纽蛋白的相对分子质量为145,000。Meta-vinculin有两种异构体pI 5.9和6.2，在体内被磷酸化，比纽蛋白高8倍。在平滑肌蛋白的免疫印迹上，[125I]Meta-vinculin与Talin以及180、150、95、70、68和45kD的未知多肽特异结合。在二维肽图上，碘标记的纽蛋白和偏纽蛋白至少有95%的主要胰凝乳蛋白酶多肽是共同的，但每种蛋白质也至少有一个似乎是唯一的高标记多肽。高盐可溶的Meta-vinculin和低盐可溶的152-kD蛋白的比较肽图(由Feramisco，J.R.，J.E.Smart，K.Burbridge，D.Helfman和G.P.Thomas，1982，J.Biol描述。化学，257：11024-11031)显示了纽蛋白样蛋白之间的广泛相似性，但暗示缺乏完全的同一性。体内脉冲追逐实验表明，偏纽蛋白和纽蛋白之间不存在前体-产物关系。纽蛋白和偏纽蛋白在生化和结构上的差异表明，偏纽蛋白在平滑肌中具有独特的功能。
Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta- vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta- vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta- vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.