A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification.
A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification.
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发表时间：
1986-01
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通讯作者：
K. Gardner;V. Bennett
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作者：
K. Gardner;V. Bennett
一种新的结合钙调素的蛋白质已被确定和纯化，从Triton X-100-不溶性残留的人红细胞血影膜（细胞骨架），通过DEAE色谱和制备速率区带蔗糖梯度沉降大于95%的同质性。这种幽灵钙调素结合蛋白是一种α/β异源二聚体，亚基Mr = 103，000（α）和97，000（β）。该蛋白质的斯托克斯半径为6.9 nm，沉降系数为6.8 S，对应于197，000的分子量。此外，该蛋白质通过Cu 2 +/菲咯啉交联成Mr = 200，000的二聚体。Mr = 97，000的β亚基通过125 I-叠氮钙调蛋白的光亲和标记被鉴定为钙调蛋白结合位点。通过用未修饰的钙调蛋白置换两种不同浓度的125 I-叠氮钙调蛋白并随后进行狄克逊图分析，估计对钙调蛋白的230 nM亲和力。这种钙调素结合蛋白以30，000拷贝/细胞存在于红细胞中，并且仅与膜相关。它与红细胞细胞骨架上的一个位点紧密结合，并完全溶解在红细胞血影膜的低离子强度提取物中。通过旋转阴影、负染色和单向阴影在电子显微镜下观察钙调素结合蛋白，表明它是一个直径为12.4 nm、高度为5.4 nm的扁平环状分子。针对钙调素结合蛋白的亲和纯化抗体鉴定了脑膜中的交叉反应Mr = 100，000的多肽。
A new protein that binds calmodulin has been identified and purified to greater than 95% homogeneity from the Triton X-100-insoluble residue of human erythrocyte ghost membranes (cytoskeletons) by DEAE chromatography and preparative rate zonal sucrose gradient sedimentation. This ghost calmodulin-binding protein is an alpha/beta heterodimer with subunits of Mr = 103,000 (alpha) and 97,000 (beta). The protein exhibits a Stokes radius of 6.9 nm and a sedimentation coefficient of 6.8 S, corresponding to a molecular weight of 197,000. Moreover, the protein is cross-linked by Cu2+/phenanthroline to a dimer of Mr = 200,000. The Mr = 97,000 beta subunit was identified as the calmodulin-binding site by photoaffinity labeling with 125I-azidocalmodulin. A 230 nM affinity for calmodulin was estimated by displacement of two different concentrations of the 125I-azidocalmodulin with unmodified calmodulin and subsequent Dixon plot analysis. This calmodulin-binding protein is present in erythrocytes at 30,000 copies/cell and is associated exclusively with the membrane. It is tightly bound to a site on red cell cytoskeletons and is totally solubilized in the low ionic strength extract derived from red cell ghost membranes. Visualization of this calmodulin-binding protein in the electron microscope by rotary shadowing, negative staining, and unidirectional shadowing indicates that it is a flattened circular molecule with a 12.4-nm diameter and a 5.4-nm height. Affinity-purified antibodies against the calmodulin-binding protein identify a cross-reacting Mr = 100,000 polypeptide(s) in brain membranes.