Klodos I, Post R L, Forbush B
Institute of Biophysics, University of Aarhus, Denmark.
J Biol Chem. 1994 Jan 21;269(3):1734-43.
Interconversion of phosphoenzyme resistant to K+ and sensitive to ADP (E1P) and phosphoenzyme resistant to ADP and sensitive to K+ (E2P) was studied in bovine brain and dog and pig kidney. The kinetics of dephosphorylation were observed by chasing phosphoenzyme formed from [32P]ATP with unlabeled ATP with or without ADP or K+. Phosphorylation in 0.6-1.0 M NaCl produced mostly ADP-sensitive potassium-insensitive E1P. A potassium chase of this phosphoenzyme exposed its rate of conversion to potassium-sensitive ADP-insensitive E2P. At 20 degrees C the rate constant was approximately 1 s-1. Simultaneous dilution of [NaCl] in the chase to 100 mM increased the constant to approximately 60 s-1, which probably qualifies E1P as an intermediate in Na,K-ATPase activity. Anions inhibited conversion according to a Hofmeister series. Na+ had no specific effect. At 0 degrees C the rate constant was < 0.4 s-1, but downward jumps in [salt] produced an acceleration to > 1 s-1 for < 3 s followed by a return to the slow rate. The rapid rate would qualify E1P to participate in Na,K-ATPase activity if this rapid state participates in the normal reaction cycle. Phosphorylation in 0.02-0.1 M NaCl produced mostly E2P. Upward jumps in [NaCl] converted E2P to E1P equally rapidly and transiently. Oligomycin and high [salt] cooperated in stabilizing E1P. Jumps in [salt] greatly and transiently increased the rate of conversion of one form of the phosphoenzyme to the other. This extraordinary result required heterogeneous kinetics. A model is proposed based on control of enzyme conformation by changes in separate unmixed phases of the lipid of the membrane.
对牛脑、狗肾和猪肾中对钾离子有抗性且对二磷酸腺苷（ADP）敏感的磷酸化酶（E1P）与对ADP有抗性且对钾离子敏感的磷酸化酶（E2P）之间的相互转化进行了研究。通过用未标记的ATP（添加或不添加ADP或钾离子）追踪由[32P]ATP形成的磷酸化酶来观察去磷酸化动力学。在0.6 - 1.0 M氯化钠中进行磷酸化主要产生对ADP敏感但对钾离子不敏感的E1P。对这种磷酸化酶进行钾离子追踪揭示了其转化为对钾离子敏感但对ADP不敏感的E2P的速率。在20℃时，速率常数约为1 s-1。在追踪过程中将[氯化钠]同时稀释至100 mM会使该常数增加至约60 s-1，这可能使E1P成为钠钾ATP酶活性的一种中间体。阴离子根据霍夫迈斯特序列抑制转化。钠离子没有特定影响。在0℃时，速率常数<0.4 s-1，但[盐]浓度的突然降低会导致在<3 s内加速至>1 s-1，随后又回到缓慢速率。如果这种快速状态参与正常反应循环，那么快速速率将使E1P有资格参与钠钾ATP酶活性。在0.02 - 0.1 M氯化钠中进行磷酸化主要产生E2P。[氯化钠]浓度的突然升高会同样迅速且短暂地将E2P转化为E1P。寡霉素和高[盐]协同作用使E1P稳定。[盐]浓度的突然变化极大且短暂地增加了一种磷酸化酶形式向另一种形式的转化速率。这一非凡结果需要非均相动力学。基于膜脂质单独未混合相的变化对酶构象的控制提出了一个模型。
