Crystals of tropomyosin and native tropomyosin.
Crystals of tropomyosin and native tropomyosin.
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原肌球蛋白和天然原肌球蛋白的晶体。
DOI：
10.1016/0022-2836(68)90190-3
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发表时间：
1968
影响因子：
5.6
通讯作者：
T. Ooi
中科院分区：
文献类型：
--
作者：
S. Higashi;T. Ooi
许多球状蛋白质在合适的条件下已经结晶，而贝利（1948）发现的原肌球蛋白可能是迄今为止唯一一种可以制成三维晶体的纤维蛋白。最近开发的x射线分析技术可以应用于这种晶体，以解决原肌球蛋白分子的结构，就像已经成功地用于几种球状蛋白一样（pen&et al., 1960; Kendrew, 1962; Phillips, 1967; Kartha, Bellow & Harker, 1967）。另一种鉴定蛋白质分子的方法，即电子显微镜，可用于阐明原肌球蛋白的晶体结构。霍奇（Hodge, 1969）和赫胥黎（Huxley, 1963）沿着这条思路进行的研究表明，纤维蛋白分子（Pen@; et al., 1966）在晶体中排列成网状结构。最近发现了一种类似原肌球蛋白的蛋白，即天然原肌球蛋白，其蛋白由原肌球蛋白和肌钙蛋白组成（Ebltshi; 1963; Ebashi & Kuroda, 1966）。这种蛋白质可以在类似于原肌球蛋白结晶所需的条件下结晶，如下所述。有趣的是，Bailey在他的论文（1948）中指出，“作为一种纯化过程，结晶是相当不充分的，因为在结晶三次的制备中仍可能检测到含色氨酸的蛋白质”，而肌钙蛋白确实含有色氨酸残基（Ebashi， S., personal communication）。本文报道了原肌凝蛋白和天然原肌凝蛋白' ~晶体的简要结果。从丙酮干燥的骨骼肌（兔）中提取原肌球蛋白，在1 M-KCl存在下，通过等电沉淀和硫酸铵分离的反复循环纯化（Kominz, Saad t L&i, 1957），直到溶液的紫外光谱给出典型的酪氨酸吸收曲线（Ooi, 1967），因为原肌球蛋白不含色氨酸残基（Bailey, 1948； Kominz等人，1967）。原肌凝蛋白和肌钙蛋白采用Ebashi的方法制备（Ebashi & Ebashi, 1964; Ebashi， S., personal communication）。实验选择的结晶条件为：O-2 m - kc和O-01~-乙酸钠缓冲液（pH 5.6）和O-2~-乙酸铵（pH 6.9）制备原肌球蛋白，0.36 M-KCl、O-01~-乙酸钠助剂（pH 6.3）制备原肌球蛋白。当pH升高时，原肌球蛋白结晶的pH值达到临界，而当pH降低时则不达到临界。天然原肌球蛋白的最佳pH值也与肌钙蛋白的含量有关，肌钙蛋白含量越小，结晶的最佳pH值越高。晶体在透析液中生长，在寒冷的房间中生长两天或更长时间。
Many globular proteins have been crystallized under suitable conditions, whereas tropomyosin, found by Bailey (1948), is probably the only fibrous protein from which three-dimensional crystals have been made so far. Techniques recently developed for X-ray analysis can be applied to this crystal to solve the structure of a tropomyosin molecule, as has been done successfully for several globular proteins (Pen& et al., 1960; Kendrew, 1962; Phillips, 1967; Kartha, Bellow & Harker, 1967). Another method of identifying protein molecules, namely by electron microscopy can be used for elucidation of the crystal structure of tropomyosin. Work along this line by Hodge (1969) and by Huxley (1963) has shown that molecules of fibrous proteins (Pen@; et al., 1966) are arranged so as to form a network structure in the crystal. Recently, a protein similar to tropomyosin, native tropomyosin, was discovered, the protein of whioh consists of tropomyosin and troponin (Ebltshi; 1963; Ebashi & Kuroda, 1966). This protein can be crystallized under conditions similar to those required for the crystallization of tropomyosin as described below. It is interesting to note that Bailey oommented in his paper (1948) that “as a purification process, crystallization is quite inadequate, since tryptophan-containing protein may still be detected in preparations three times crystallized”, and troponin does contain tryptophan residues (Ebashi, S., personal communication). In the present communication, brief results on’~ crystals of both tropomyosin and native tropomyosin are reported.Tropomyosin extracted from acetone-dried skeletal muscle (rabbit) was purified by repeated oycles of isoelectric precipitation and ammonium sulphate separation in the presence of 1 M-KCl (Kominz, Saad t L&i, 1957) until the ultraviolet spectrum of solutions gave a typical absorption curve of tyrosine (Ooi, 1967), since tropomyosin does not contain tryptophctn residues (Bailey, 1948; Kominz et al., 1967). Native tropomyosin and troponin were prepared by Ebashi’s procedure (Ebashi & Ebashi, 1964; Ebashi, S., personal communication). The following crystallization conditions were chosen empirically: O-2 M-KC& O-01~-sodium aceMe buffer (pH 5.6) and O-2~-ammonium acetate (pH 6.9) for tropomyosin, and 0.36 M-KCl, O-01~-sodium acetate buiIer (pH 6.3) for native tropomyosin. The pH values for crystallization of tropomyosin were critical when the pH was raised but not when the pH was lowered. Also the optimal pH for native tropomyosin was dependent on the content of troponin-the smaller the troponin content the higher the optimal pH for crystallization. Crystals were grown in a dialysis seek for two days or longer in a cold room.