Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange.
Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange.
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DOI：
10.1021/bi00154a012
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发表时间：
1992-10
期刊：
影响因子：
2.9
通讯作者：
S. Sadis;L. Hightower
中科院分区：
文献类型：
--
作者：
S. Sadis;L. Hightower
哺乳动物70千道尔顿热休克同源蛋白（Hsc 70）是一种丰富的细胞溶质分子伴侣，其与蛋白质底物的相互作用受ATP水解调节。在体外，纯化的Hsc 70被发现有一个缓慢的，内在的ATP酶活性的蛋白质底物的情况下。加入未折叠的蛋白质如脱辅基细胞色素c刺激ATP水解2-3倍。与此相反，本机holoprotein，细胞色素c，没有刺激ATP酶的速率，在雅阁与最近的观察，70千道尔顿热休克蛋白选择性地与未折叠的蛋白质相互作用。脱辅基细胞色素c刺激ATP水解是由于Vmax的增加，对ATP的Km没有影响。[3 H]ATP水解后，形成相对稳定的[3 H]ADP.Hsc70复合物。Hsc 70的[3 H]ADP释放在其他核苷酸如ADP或ATP存在下最有效，表明ADP释放作为ADP/ATP交换反应发生。在外源核苷酸存在下，Hsc 70的放射性标记ADP的损失遵循一级动力学。在核苷酸的存在下，脱辅基细胞色素c诱导的ADP从Hsc 70释放的速率增加了2倍。此外，在无和存在脱辅基细胞色素c（0.16和0.34分钟-1，分别）的情况下测量的核苷酸交换反应的速率常数密切匹配的kcat值来自ATP水解测量（0.15和0.38分钟-1，分别）。结果表明，ADP的释放在Hsc 70 ATP酶反应的限速步骤和未折叠的蛋白质刺激ATP水解加快ADP/ATP交换的速率。
The mammalian 70-kilodalton heat shock cognate protein (Hsc70) is an abundant, cytosolic molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis. In vitro, purified Hsc70 was found to have a slow, intrinsic ATPase activity in the absence of protein substrates. The addition of an unfolded protein such as apocytochrome c stimulated ATP hydrolysis 2-3-fold. In contrast, the native holoprotein, cytochrome c, did not stimulate the ATPase rate, in accord with recent observations that 70-kilodalton heat shock proteins interact selectively with unfolded proteins. Stimulation of ATP hydrolysis by apocytochrome c was due to an increase in the Vmax, with no effect on the Km for ATP. Following hydrolysis of [3H]ATP, a relatively stable [3H]ADP.Hsc70 complex was formed. Release of [3H]ADP from Hsc70 was most efficient in the presence of other nucleotides such as ADP or ATP, suggesting that ADP release occurs as an ADP/ATP exchange reaction. The loss of radiolabeled ADP from Hsc70 in the presence of exogenous nucleotides followed first-order kinetics. In the presence of nucleotides, apocytochrome c induced a 2-fold increase in the rate of ADP release from Hsc70. Moreover, rate constants of the nucleotide exchange reaction measured in the absence and presence of apocytochrome c (0.16 and 0.34 min-1, respectively) closely matched the kcat values derived from ATP hydrolysis measurements (0.15 and 0.38 min-1, respectively). The results suggest that ADP release in a rate-limiting step in the Hsc70 ATPase reaction and that unfolded proteins stimulate ATP hydrolysis by accelerating the rate of ADP/ATP exchange.