Cornelius Flemming, Turner Nigel, Christensen Hanne R Z
Department of Biophysics, University of Aarhus, Ole Worms Allé 185, DK-8000, Denmark.
Biochemistry. 2003 Jul 22;42(28):8541-9. doi: 10.1021/bi034532e.
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) - P --> 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.
本文描述了磷脂酰链长度（n(c)）和胆固醇对重构于特定脂质组成脂质体中的钠钾ATP酶几个部分反应的影响。这涉及E(1)/E(2)平衡、磷酸化酶水平以及K(+)解封闭反应。此外，还研究了脂质对一些稳态性质的影响。最后，研究了胆固醇对磷酸化和自发去磷酸化反应温度敏感性的影响。天然钠钾ATP酶膜制剂的脂肪酸和胆固醇组成与已知支持最大水解能力的脂质组成（由体外实验测定）显示出显著相似性。钠钾ATP酶反应的主要限速步骤，即E(2)→E(1)反应，以及其他几个部分反应都被胆固醇加速。这涉及ATP的磷酸化以及E(1)-P→E(2)-P反应。此外，胆固醇使E(1)/E(2)平衡向E(1)构象移动，并提高了K(+)解封闭速率。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链长度所诱导的作用，这表明胆固醇的作用并非完全由增加疏水双层厚度引起，这暗示了对钠钾ATP酶的另一种作用机制。
