Characterization of a second myosin from Acanthamoeba castellanii.
Characterization of a second myosin from Acanthamoeba castellanii.
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来自卡斯氏棘阿米巴的第二种肌球蛋白的表征。
DOI：
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发表时间：
1978
影响因子：
4.8
通讯作者：
M. Porter
中科院分区：
文献类型：
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作者：
T. Pollard;W. Stafford;M. Porter
我们从卡氏棘阿米巴中纯化出了分子量为 400,000 的肌球蛋白，即肌球蛋白-II。经过离子交换层析、肌动球蛋白沉淀、肌动蛋白提取和凝胶渗透层析的顺序，每 100 g 细胞产生约 11 mg 肌球蛋白-II，纯度为 90% 至 96%。 ATP 酶活性在存在 Ca2+ 的情况下最高，但该酶在存在高浓度 K+ 的 EDTA 中也具有活性。该分子由两条175,000分子量重链、一条或两条17,500分子量轻链和两条16,500分子量轻链组成。肌球蛋白-II富含酸性残基，每摩尔含有约32个半胱氨酸残基。沉降系数为5.9S。特性粘度为126cc/g。通过平衡超速离心，平均分子量取决于初始上样浓度，表明 400,000 分子量物质与 200,000 分子量物质处于平衡状态。通过电子显微镜观察，该分子在 90 nm 长的尾部一端有两个球状头部。在小于 0.25 M 的 KCl 溶液中，肌球蛋白-II 尾部自缔合形成非常小的 (6.6 x 205 nm) 双极丝的主链，中心裸露区长 97 nm。肌球蛋白-II 与肌动蛋白丝结合，形成周期性箭头形复合物，但其 Mg2+ ATP 酶活性仅被肌动蛋白激活 50% 或更少。当放射性肌球蛋白-II 在未标记的棘阿米巴匀浆中孵育长达 90 分钟时，它不会降解成更小的片段，例如分子量为 190,000 的肌球蛋白-I。我们的观察和 Maruta 和 Korn ((1977) J. Biol. Chem. 252, 6501-6509) 提供的详细酶学数据认为，较小的棘阿米巴肌球蛋白-I (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem, 248, 4682-2690) 并不是由匀浆或提取物中的肌球蛋白-II。
We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii. The sequence of ion exchange chromatography, actomyosin precipitation, actin extraction, and gel permeation chromatography yields per 100 g of cells about 11 mg of myosin-II which is 90 to 96% pure. ATPase activity is highest in the presence of Ca2+, but the enzyme is also active in EDTA provided high concentrations of K+ are present. The molecule consists of two 175,000 molecular weight heavy chains, one or two 17,500 molecular weight light chains, and two 16,500 molecular weight light chains. Myosin-II is rich in acidic residues and contains about 32 residues of cysteine/mol. The sedimentation coefficient is 5.9 S. Intrinsic viscosity is 126 cc/g. By equilibrium ultracentrifugation, the molecular weight averages depended upon the initial loading concentration in a way that suggested a 400,000 molecular weight species is in equilibrium with a 200,000 molecular weight species. By electron microscopy the molecule was seen to have two globular heads at one end of a tail 90 nm long. In KCl solutions of less than 0.25 M, the myosin-II tails self-associate to form the backbone of very small (6.6 x 205 nm) bipolar filaments with central bare zones 97 nm long. Myosin-II binds to actin filaments, forming periodic arrowhead-shaped complexes, but its Mg2+ ATPase activity is activated only 50% or less by actin. When radioactive myosin-II is incubated up to 90 min in unlabeled Acanthamoeba homogenates, it is not degraded into smaller fragments, such as the 190,000 molecular weight myosin-I. Our observations and the detailed enzymatic data presented by Maruta and Korn ((1977) J. Biol. Chem. 252, 6501-6509) argue that the smaller Acanthamoeba myosin-I (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem, 248, 4682-2690) does not arise by fragmentation of myosin-II in the homogenate or extract.