Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium.
Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium.
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DOI：
10.1083/jcb.85.2.361
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发表时间：
1980-05
期刊：
影响因子：
--
通讯作者：
Taylor DL
中科院分区：
文献类型：
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作者：
Fowler V;Taylor DL
当向由人红细胞膜制备的低盐提取物中添加纯化的兔肌肉 G-或 F-肌动蛋白以得到约 1 mg/ml 的浓度时，会形成固体凝胶。该提取物含有血影蛋白、肌动蛋白、条带 4.1、条带 4.9、血红蛋白和几种微量成分。将凝胶材料在 43,000 g 下离心 10 分钟获得的沉淀含有血影蛋白、肌动蛋白、条带 4.1 和条带 4.9。尽管在添加肌动蛋白时，稀释数倍的提取物不会胶凝，但与同等浓度的单独的 G-肌动蛋白、单独的提取物或单独的 F-肌动蛋白相比，混合物的粘度显着增加。热变性提取物完全失去活性。在生理离子强度和pH条件下，通过将游离钙离子浓度提高到微摩尔水平，这种超分子结构的信息受到抑制。通过在 37 摄氏度下初始提取（并在 0 摄氏度下保存）制备的低盐提取物仅在加热后添加肌动蛋白凝胶，而通过在 0 摄氏度下提取制备的提取物在冰上和加热后都具有活性。在有利于血影蛋白二聚体转化为四聚体的条件下预温育 37 摄氏度低盐提取物可大大增强 0 摄氏度下的凝胶活性。相反，在有利于血影蛋白四聚体转化为二聚体的条件下预温育 0 摄氏度低盐提取物会大大降低 0 摄氏度下的凝胶活性。从 37 度或 0 摄氏度纯化血影蛋白二聚体或四聚体低盐提取物，通过琼脂糖凝胶 4B 在 4 摄氏度下进行凝胶过滤。将肌动蛋白添加到纯化的血影蛋白二聚体（32℃）或四聚体（0℃或32℃）中会导致粘度相对较小的增加，而将肌动蛋白添加到包含血影蛋白、肌动蛋白、条带4.1和条带4.9的高分子量复合物（HMW复合物）中会导致粘度急剧增加，对钙敏感。这些粘度与 37 摄氏度或 0 摄氏度低盐提取物获得的粘度相当。将纯化的条带 4.1 添加到纯化的血影蛋白二聚体（32 摄氏度）或纯化的血影蛋白四聚体（0 摄氏度）加肌动蛋白中，会导致粘度大幅增加，与 HMW 复合物和粗提取物中观察到的结果类似，这与 E. Ungewickell、P. M. Bennett、R. Calvert、V. Ohanian 和 W. B. Gratzer 最近的报告一致。 1979 年《自然》（伦敦）280：811-814。我们认为这种血影蛋白-肌动蛋白带 4.1 凝胶代表了红细胞细胞骨架的主要结构成分。
A low-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G- or F-actin is added to it to give a concentration of approximately 1 mg/ml. This extract contains spectrin, actin, band 4.1, band 4.9, hemoglobin, and several minor components. Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9. Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations. Heat-denatured extract is completely inactive. Under conditions of physiological ionic strength and pH, information of this supramolecular structure is inhibited by raising the free calcium ion concentration to micromolar levels. Low-salt extracts prepared by initial extraction at 37 degrees C (and stored at 0 degree C) gel after actin is added to them only when warmed, whereas extracts prepared by extraction at 0 degree C are active on ice as well as after warming. Preincubation of the 37 degrees C low-salt extract under conditions that favor conversion of spectrin dimer to tetramer greatly enhances gelation activity at 0 degree C. Conversely, preincubation of the 0 degree C low-salt extract under conditions that favor conversion of spectrin tetramer to dimer greatly diminishes gelation activity at 0 degree C. Spectrin dimers or tetramers are purified from the 37 dgrees or 0 degree C low-salt extract by gel filtration at 4 degrees C over Sepharose 4B. The addition of actin to either purified spectrin dimer (at 32 degrees C) or tetramer (at 0 degree C or 32 degrees C) results in relatively small increases in viscosity, whereas the addition of actin to a high-molecular-weight complex (HMW complex) containing spectrin, actin, band 4.1, and band 4.9 results in dramatic, calcium-sensitive increases in viscosity. These viscosities are comparable to those obtained with the 37 degrees or 0 degree C low-salt extracts. The addition of purified band 4.1 to either purified spectrin dimer (at 32 degrees C) or purified spectrin tetramer (at 0 degree C) plus actin results in large increases in viscosity similar to those observed for the HMW complex and the crude extract, which is in agreement with a recent report by E. Ungewickell, P. M. Bennett, R. Calvert, V. Ohanian, and W. B. Gratzer. 1979 Nature (Lond.) 280:811-814. We suggest that this spectrin-actin-band 4.1 gel represents a major structural component of the erythrocyte cytoskeleton.