Johnson R P, Craig S W
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Biochem Biophys Res Commun. 1995 May 5;210(1):159-64. doi: 10.1006/bbrc.1995.1641.
Using a gel filtration assay, we have characterized the binding of acidic phospholipids to vinculin. Vinculin binds phosphatidyinositol (Kd approximately 5 microM) in a reversible, saturable manner in low ionic strength buffers. This interaction is inhibited substantially at 100 mM NaCl and therefore may not be of physiological interest. In contrast, the carboxy-terminal 30-kDa fragment of vinculin, produced by S. aureus V8 protease cleavage, binds acidic phospholipids more tightly than the intact protein, and in a manner insensitive to 100 mM NaCl. Re-addition of the 95-kDa head fragment to the tail restores salt-sensitivity to the tail-lipid interaction. These data indicate that under physiologic ionic conditions, the intramolecular head-tail interaction in vinculin masks a high affinity acidic phospholipid binding site present in the tail domain.
利用凝胶过滤分析，我们已对酸性磷脂与纽蛋白的结合特性进行了表征。在低离子强度缓冲液中，纽蛋白以可逆、饱和的方式结合磷脂酰肌醇（解离常数约为5微摩尔）。这种相互作用在100毫摩尔氯化钠浓度下会受到显著抑制，因此可能没有生理意义。相比之下，经金黄色葡萄球菌V8蛋白酶切割产生的纽蛋白羧基末端30千道尔顿片段比完整蛋白更紧密地结合酸性磷脂，且对100毫摩尔氯化钠不敏感。将95千道尔顿的头部片段重新添加到尾部可恢复尾部与脂质相互作用的盐敏感性。这些数据表明，在生理离子条件下，纽蛋白分子内的头尾相互作用掩盖了尾部结构域中存在的高亲和力酸性磷脂结合位点。
