Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes.
Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes.
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DOI：
10.1016/s0021-9258(18)45276-3
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发表时间：
1987-09
期刊：
影响因子：
--
通讯作者：
V. Fowler
中科院分区：
文献类型：
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作者：
V. Fowler
125 I标记的Bolton-Hunter原肌球蛋白与经十二烷基硫酸钠凝胶电泳分离的膜蛋白的硝酸纤维素印迹结合，在红细胞膜上鉴定出一种新的Mr 43，000的原肌球蛋白结合蛋白（TMBP）。这种蛋白质不是肌动蛋白，因为125 I-原肌球蛋白在印迹上不与纯化的肌动蛋白结合。125 I-原肌球蛋白与这种蛋白质的结合是特异性的，因为它被过量的未标记的原肌球蛋白抑制，但不被F-肌动蛋白或肌钙蛋白抑制。这种蛋白质已被纯化到95%的同质性，从1 M的Tris提取物的原肌球蛋白耗尽红细胞膜的DEAE-纤维素和羟基磷灰石层析，然后通过凝胶过滤Ultrogel AcA 44。纯化的蛋白质具有3.9 nm的斯托克斯半径和2.8 S的沉降系数，对应于43，000的天然分子量。125 I-原肌球蛋白与纯化的TMBP的结合在一个原肌球蛋白分子（Mr 60，000）到两个Mr 43，000的TMBP时饱和，亲和力约为5 × 10（−7）M。在用非离子去污剂Triton X-100提取膜后，TMBP与膜骨架结合，并且相对于原肌球蛋白以每两个原肌球蛋白分子约一个的比例存在。由于有足够的原肌球蛋白，使两个原肌球蛋白分子与膜骨架中的每一个短肌动蛋白丝相关联，红细胞膜TMBP与原肌球蛋白一起，可以起到限制附着在每一个短肌动蛋白丝上的血影蛋白分子数量的作用，从而指定血影蛋白-肌动蛋白晶格的六边形对称性。或者，这种TMBP可能是同源的肌肉肌钙蛋白之一，并可能与原肌球蛋白调节红细胞肌动球蛋白-ATP酶的活性和影响红细胞的形状。
A new Mr 43,000 tropomyosin-binding protein (TMBP) has been identified in erythrocyte membranes by binding of 125I-labeled Bolton-Hunter tropomyosin to nitrocellulose blots of membrane proteins separated by sodium dodecyl sulfate-gel electrophoresis. This protein is not actin, because 125I-tropomyosin does not bind to purified actin on blots. Binding of 125I-tropomyosin to this protein is specific because it is inhibited by excess unlabeled tropomyosin but not by F-actin or muscle troponins. This protein has been purified to 95% homogeneity from a 1 M Tris extract of tropomyosin-depleted erythrocyte membranes by DEAE-cellulose and hydroxylapatite chromatography, followed by gel filtration on Ultrogel AcA 44. The purified protein has a Stokes radius of 3.9 nm and a sedimentation coefficient of 2.8 S, corresponding to a native molecular weight of 43,000. Binding of 125I-tropomyosin to the purified TMBP saturates at one tropomyosin molecule (Mr 60,000) to two Mr 43,000 TMBPs, with an affinity of about 5 × 10(−7) M. The TMBP is associated with the membrane skeleton after extraction of membranes with the non-ionic detergent, Triton X-100, and is present with respect to tropomyosin at a ratio of about one for every two tropomyosin molecules. Because there is enough tropomyosin for two tropomyosin molecules to be associated with each of the short actin filaments in the membrane skeleton, the erythrocyte membrane TMBP, together with tropomyosin, could function to restrict the number of spectrin molecules attached to each of the short actin filaments and thus specify the hexagonal symmetry of the spectrin-actin lattice. Alternatively, this TMBP could be homologous to one of the muscle troponins and might function with tropomyosin to regulate erythrocyte actomyosin-ATPase activity and influence erythrocyte shape.