Yoda A, Yoda S
J Biol Chem. 1987 Jan 5;262(1):110-5.
The phosphorylated intermediate (EP) of the Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme is composed of an ADP-sensitive K+-insensitive form (E1P), an ADP- and K+-sensitive form (EP), and a K+-sensitive ADP-insensitive form (E2P). The composition of the intermediate varied with the cholesterol content of the lipid bilayer. The PL containing less than 30 mol % cholesterol (LCPL) formed E2P-rich EP in the presence of 10 mM Na+ on both sides at 15 degrees C, while the PL containing more than 35 mol % cholesterol (HCPL) formed EP-rich EP under the same condition. In the presence of ionophore (monensin, nigericin, A23187), the HCPL formed E2P-rich EP as reported in the preceding paper. The turnover rate of Na-ATPase activity (the ratio of Na-ATPase to the EP level) in the LCPL was lower than that in the HCPL, and the addition of 20 microM monensin or A23187 to the HCPL reduced the Na-ATPase activity. The coupling ratio of Na+ influx (cellular efflux):Na+ efflux (cellular influx):ATP hydrolysis was 2.8:1.8:1 in the LCPL, although 1.6:0.6:1 in the HCPL. The coupling ratio of Na+ influx:ATP hydrolysis in the HCPL increased to 2.8:1 in the presence of A23187. Moreover, the increase of ATP concentration enhanced not only the Na-ATPase activity in the LCPL and HCPL with monensin but also the Na+ influx in the LCPL. This ATP enhancement was not found, however, in the HCPL without ionophores. The ADP enhancement of the Na+ influx was not observed in either the HCPL or the LCPL. We conclude from these observations that there are at least two different phosphorylation-dephosphorylation cycles (an E2P cycle and an EP cycle) in the PL in the absence of K+. The E2P cycle transports three Na+ from the extravesicular (cytoplasmic) to the intravesicular (extracellular) side and two Na+ in the opposite direction per cycle and is similar to the ATP-dependent Na+-Na+ exchange system already reported (Blostein, R. (1983) J. Biol. Chem. 258, 7948-7953; Cornelius, F., and Skou, J. C. (1985) Biochim. Biophys. Acta 818, 211-221). However, the EP cycle transports one Na+ from the extravesicular to the intravesicular side/cycle and has not yet been previously reported.
由电鳗酶制备的钠钾ATP酶蛋白脂质体（PL）的磷酸化中间体（EP）由对ADP敏感、对K⁺不敏感的形式（E1P）、对ADP和K⁺敏感的形式（EP）以及对K⁺敏感、对ADP不敏感的形式（E2P）组成。中间体的组成随脂质双层中胆固醇含量的变化而变化。胆固醇含量低于30摩尔%的PL（LCPL）在15℃两侧均存在10 mM Na⁺的情况下形成富含E2P的EP，而胆固醇含量超过35摩尔%的PL（HCPL）在相同条件下形成富含EP的EP。在离子载体（莫能菌素、尼日利亚菌素、A23187）存在的情况下，HCPL如前一篇论文所报道的那样形成富含E2P的EP。LCPL中钠ATP酶活性的周转率（钠ATP酶与EP水平的比率）低于HCPL，并且向HCPL中添加20 μM莫能菌素或A23187会降低钠ATP酶活性。LCPL中Na⁺内流（细胞外流）：Na⁺外流（细胞内流）：ATP水解的偶联比率为2.8:1.8:1，而HCPL中为1.6:0.6:1。在A23187存在的情况下，HCPL中Na⁺内流：ATP水解的偶联比率增加到2.8:1。此外，ATP浓度的增加不仅增强了添加莫能菌素的LCPL和HCPL中的钠ATP酶活性，还增强了LCPL中的Na⁺内流。然而，在没有离子载体的HCPL中未发现这种ATP增强作用。在HCPL或LCPL中均未观察到ADP对Na⁺内流的增强作用。我们从这些观察结果得出结论，在不存在K⁺的情况下，PL中至少存在两个不同的磷酸化 - 去磷酸化循环（一个E2P循环和一个EP循环）。E2P循环每个循环将三个Na⁺从囊泡外（细胞质）转运到囊泡内（细胞外）侧，并将两个Na⁺以相反方向转运，这与已经报道的ATP依赖性Na⁺ - Na⁺交换系统相似（Blostein，R.（1983）J. Biol. Chem. 258，7948 - 7953；Cornelius，F.，和Skou，J. C.（1985）Biochim. Biophys. Acta 818，211 - 221）。然而，EP循环每个循环将一个Na⁺从囊泡外转运到囊泡内侧，这是以前尚未报道过的。
