Modulation of Na,K-ATPase by phospholipids and cholesterol. II. Steady-state and presteady-state kinetics
Modulation of Na,K-ATPase by phospholipids and cholesterol. II. Steady-state and presteady-state kinetics
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DOI：
10.1021/bi034532e
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发表时间：
2003-07-22
期刊：
影响因子：
2.9
通讯作者：
Christensen, HRZ
中科院分区：
文献类型：
--
作者：
Cornelius, F;Turner, N;Christensen, HRZ
描述了磷脂酰基链长(n(C))和胆固醇对Na，K-ATPase重组为特定脂组成的脂质体的几个部分反应的影响。这与E-1/E-2平衡、磷酸酶水平和K+解锁反应有关。此外，还考察了脂类对某些稳态性质的影响。最后，研究了胆固醇对磷酸化反应和自发去磷酸化反应的温度敏感性的影响。天然Na，K-ATPase膜制剂的脂肪酸和胆固醇组成与体外实验所确定的支持最大水解力的脂类组成显著相似。胆固醇加速了Na，K-ATPase反应、E-2->；E-1反应以及其他几个部分反应的主要速率决定步骤。这涉及到ATP的磷酸化以及E(1)类似的top-gt；E-2-P反应。此外，胆固醇使E-1/E-2平衡向E-1构象移动，增加了K+-解闭塞率。最后，胆固醇显著影响自发去磷酸化反应和ATP磷酸化的温度敏感性。胆固醇的作用并不完全等同于增加磷脂酰基的长度，这表明胆固醇的作用并不完全是由增加疏水双层厚度引起的，这表明了对Na，K-ATPase的另一种作用机制。
The effects of phospholipid acyl chain length (n(c)) and cholesterol on several partial reactions of Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. This regards the E-1/E-2 equilibrium, the phosphoenzyme level, and the K+-deocclusion reaction. In addition, the lipid effects on some steady-state properties were investigated. Finally, the effects of cholesterol on the temperature sensitivity of the phosphorylation and spontaneous dephosphorylation reactions were investigated. The fatty acid and cholesterol composition of the native Na,K-ATPase membrane preparation showed a remarkable similarity to the lipid composition known to support maximum hydrolytic capacity as determined from in vitro experiments. The main rate-determining step of the Na,K-ATPase reaction, the E-2 --> E-1 reaction, as well as several other partial reactions were accelerated by cholesterol. This regards the phosphorylation by ATP as well as the E(1)similar toP --> E-2-P reaction. Moreover, cholesterol shifted the E-1/E-2 equilibrium toward the E-1 conformation and increased the K+-deocclusion rate. Finally, cholesterol significantly affected the temperature sensitivity of the spontaneous dephosphorylation reaction and the phosphorylation by ATP. The effects of cholesterol were not completely equivalent to those induced by increasing the phospholipid acyl chain length, indicating that the cholesterol effects are not entirely caused by increasing the hydrophobic bilayer thickness, which indicates an additional mechanism of action on the Na,K-ATPase.