Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate.
Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate.
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使用新型磷酸盐荧光探针测量相关兔骨骼肌动球蛋白的核苷三磷酸裂解和磷酸盐释放步骤的动力学。
DOI：
10.1021/bi970540h
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Webb,MR
中科院分区：
文献类型：
--
作者：
White,HD;Belknap,B;Webb,MR
我们使用双混合停流荧光分光光度计，在非常低的离子强度下测量了肌动球蛋白核苷三磷酸（NTP）水解单次转换过程中无机磷酸盐（Pi）释放的动力学，以增加肌球蛋白-ATP和肌球蛋白-ADP-Pito肌动蛋白的亲和力。将肌球蛋白亚片段1和一系列核苷三磷酸混合并孵育101 −10 s，使NTP与肌球蛋白结合，并产生肌球蛋白−NTP和肌球蛋白−NDP−Pi的稳态混合物。然后将稳态中间体与肌动蛋白混合。基于磷酸盐结合蛋白，使用Pi的荧光探针测量Pirelease的动力学[Brune et al.（1994）Biochemistry 33，8262 - 8271]。这些数据与猝灭流数据，其中的水解的快速爆发的程度在ATP水解的第一个营业额，其次是化学猝灭的反应混合物在不同的时间后，迅速混合ATP和肌球蛋白亚片段1。根据双混合肌动球蛋白测量，Pirelease的动力学是双相的。快相对应于相关肌动球蛋白-ADP-Picomplex的Pirelease。慢相测量在相关肌动球蛋白上的裂解步骤的速率。在肌动蛋白饱和时，快相的振幅与在没有肌动蛋白的情况下通过淬灭流观察到的Piburst的大小之间存在相关性：该相的大小对应于第一次混合时肌球蛋白−ADP− Piburst的量。在20 °C下，对于ATP，Pirelease步骤的速率为75（±5）s-1，比裂解步骤大25倍，裂解步骤是肌动球蛋白ATP水解饱和肌动蛋白的限速步骤。Pirelease的速率常数仅随核苷结构略有变化。Pirelease的慢相的速率常数（测量裂解）高度依赖于NTP底物的结构。
We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin−ATP and myosin−ADP−Pito actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for ∼1−10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin−NTP and myosin−NDP−Pi. The steady state intermediates were then mixed with actin. The kinetics of Pirelease were measured using a fluorescent probe for Pi, based on a phosphate binding protein [Brune et al. (1994)Biochemistry 33, 8262−8271]. These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATP hydrolysis was followed by chemical quenching of the reaction mix at various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of Pirelease are biphasic. The fast phase corresponds to Pirelease from the associated actomyosin−ADP−Picomplex. The slow phase measures the rate of the cleavage step on associated actomyosin. At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the Piburst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin−ADP−Piformed during the first mix. For ATP at 20 °C the rate of the Pirelease step is 75 (±5) s-1, 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of Pirelease varies only slightly with nucleoside structure. The rate constant of the slow phase of the Pirelease (measuring cleavage) is highly dependent upon the structure of the NTP substrate.