Stabilization of Na,K-ATPase by ionic interactions.
Stabilization of Na,K-ATPase by ionic interactions.
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通过离子相互作用稳定 Na,K-ATP 酶。
DOI：
10.1016/j.bbamem.2007.12.006
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
M. Esmann
中科院分区：
文献类型：
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作者：
E. Fodor;N. Fedosova;Csilla Ferencz;D. Marsh;T. Páli;M. Esmann
采用差示扫描量热法(DSC)和活性测定方法，研究了离子对鲨鱼盐腺Na，K-ATPase热稳定性和去折叠的影响，并与猪肾Na，K-ATPase进行了比较。在pH为7的1 mM组氨酸中，鲨鱼酶在20℃时迅速失活，肾酶在42℃时也是如此(但在20℃时不会)。通过添加20 mM的组氨酸或1 mM的氯化钠或氯化钾来增加离子强度，可以保护两种酶免受这种快速失活的影响。差示扫描量热仪检测到，鲨鱼酶在较低温度(TM≈45°C)下经历热展开，而肾脏酶(TM≈55°C)则在较低温度下发生热去折叠。这两个量热吸热曲线都表明了多步展开，可能与不同的合作区域有关。虽然1 mM和20 mM组氨酸中的肾酶的总展开热相似，但相对于20 mM组氨酸中的组氨酸，鲨鱼酶在1 mM组氨酸中的展开转变会损失中点温度较高的成分。这归因于在低离子强度下DSC样品的离心过程中由于高静水压力而导致酶的部分展开，这与失活测量相关。在1 mM组氨酸中向鲨鱼酶中加入10 mM氯化钠可防止DSC样品在离心过程中失活，但在加入氯化钠之前在20℃孵育1小时会导致展开过渡期间中点温度较低的成分损失。因此，毫摩尔浓度的阳离子至少提供了两种不同的稳定模式，可能会影响单独的合作区域。两种Na，K-ATPase不同的热稳定性和变性温度与各自的生理温度有关，可能与不同的脂质环境有关。
The effect of ions on the thermostability and unfolding of Na,K–ATPase from shark salt gland was studied and compared with that of Na,K–ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm≈45 °C) than does the kidney enzyme (Tm≈55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K–ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.