ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.
ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.
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DOI：
10.1083/jcb.99.5.1867
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发表时间：
1984-11
期刊：
影响因子：
--
通讯作者：
Spudich JA
中科院分区：
文献类型：
--
作者：
Sheetz MP;Chasan R;Spudich JA
Sheetz和Spudich （1983, Nature (Lond.), 330:31-35）表明，可以在体外使用Nitella的肌球蛋白包被珠和定向肌动蛋白电缆来测量肌动蛋白沿着肌动蛋白丝的ATP依赖运动。为了将这种体外运动作为一种定量分析，并更好地了解运动的基础，我们定义了影响肌球蛋白头速度的因素。涂有骨骼肌肌球蛋白的珠子以2-6微米/秒的速度移动，这取决于肌球蛋白的制备。当浓度高于临界值（约20微克肌球蛋白/2.5 × 10(9)直径为1微米的微球）时，该流速与微球表面的肌球蛋白浓度无关。pH值6.8和7.5之间的运动是最优的,在氯化钾浓度小于70毫米,在ATP浓度大于0.1毫米,和Mg2 +浓度2和6毫米之间。从珠速度对温度的依赖关系,我们计算90焦每摩尔的活化能低于22度和40焦每摩尔22度以上不同肌凝蛋白C .物种的特征速度移动,这些速度正比于其actin-activated ATP酶的活动。此外，涂有平滑肌肌凝蛋白或骨骼肌肌凝蛋白的小球的速度与已知的肌凝蛋白在这些肌肉中沿着肌动蛋白细丝运动的体内速率密切相关。因此，这种体外实验提供了一种快速、可重复的方法，用于定量肌动蛋白上肌球蛋白分子的ATP依赖运动。
Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP- dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP- dependent movement of myosin molecules on actin.
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影响因子：
3.4
作者：
MOSS, RL;HAWORTH, RA
通讯作者：
HAWORTH, RA
影响因子：
3.4
作者：
STEIN, RB;GORDON, T;SHRIVER, J
通讯作者：
SHRIVER, J
影响因子：
56.9
作者：
HUXLEY, HE
通讯作者：
HUXLEY, HE
影响因子：
64.8
作者：
SHEETZ, MP;SPUDICH, JA
通讯作者：
SPUDICH, JA
DOI：
10.1101/sqb.1973.037.01.021
发表时间：
1973-01-01
期刊：
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
影响因子：
--
作者：
MOOS, C
通讯作者：
MOOS, C