Molecular Mechanism of Energy Transduction and Regulation in Na^+ pump and K^+ pump

Na^+泵和K^+泵分子机制的能量转导与调控。研究了Na^+泵中标记探针间的荧光能量转移。结果表明，从BIPM探针（cys964）到FITC探针（lys501）的荧光能量转移随NaE_1经E_1P生成E_2P而增加，随E_2P经KE_2生成NaE_1而减少。两种探针的各向异性随这些中间产物的形成变化不大。现在检测到从BIPM到ANM探针的荧光能量转移的相反变化。这些探测器之间距离的估计正在进行中。比较了Na^+泵和K^+泵的构象变化与磷酸化-去磷酸化循环的关系。这些数据表明，构象变化与磷酸化-去磷酸化循环并不是紧密耦合的，有力地表明，构象变化反映了Na^+和K^+在Na^+泵分子中的迁移。为了阐明能量转导的机制，量化这些离子的迁移似乎是必不可少的。用内源性（色氨酸）和外源性（BIPM， FITC）荧光探针研究了H^+泵的构象变化。磷酸酶形成后，色氨酸残基的荧光强度增加。在磷酸酶中加入K^+，使荧光降低到与HE_1相同的水平。BIPM探针的荧光强度随磷酸化速率常数的变化而下降。磷酸酶中加入K^+后，BIPM荧光略有增加。这些数据清楚地表明，两种荧光探针感知不同的酶状态，这已经在使用各种荧光探针的Na^+泵中显示出来。修饰Na^+泵，在Na^+或K^+存在的情况下，用磷酸吡哆醛标记ATP结合域附近的荧光探针。Na^+， K^+-ATP酶活性和ATP磷酸化被抑制，而乙酰磷酸磷酸化仍然存在。吡哆醛磷酸探针在NaE_1中的附着位点为Lys-664，在KE_2.4中的附着位点为Lys-664和Lys-469。研究了磷脂在Na^+、K^+- atp酶构象变化中的作用。结果表明，BIPM探针在Cys-964位点随E_2P积累的荧光强度变化比E_1P更容易受到磷脂酶A处理的影响。从猪肾红外髓质中纯化了环amp依赖性蛋白激酶，并纯化了Na^+， K^+- atp酶。Na^+， K^+- atp酶被纯化的激酶磷酸化。在磷酸化位点的1摩尔α链序列中检测到1摩尔磷酸，目前正在进行激酶磷酸化后Na^+， K^+- atp酶活性的变化以及对配体的亲和力的研究。少

Energy Transduction and Regulation in Molecular Mechanism of Na^+ pump and K^+ pump1. Studies of Fluorescence energy transfer between labeled probes in Na^+ pump were made during sequential appearance of reaction intermediates. The data clearly showed that the fluorescence energy transfer from BIPM probe (Cys 964) to FITC probe (Lys 501) increased accompanying formation of E_2P from NaE_1 via E_1P, and decreased accompanying formation of NaE_1 from E_2P via KE_2. The anisotropy of both probes changed little accompanying formation of these intermediates. Opposite change in fluorescence energy transfer from BIPM to ANM probes are now detected. The estimation of the distances between those probes are in progress.2. Relationships between conformational changes and phosphorylation-dephosphosphorylation cycles in Na^+ pump and K^+ pump were compared. The data show that the conformational changes and the phosphorylation-dephosphorylation cycle is not tightly coupled and strongly suggest that … More the conformational changes reflect the migration of Na^+ and K^+ in the Na^+ pump molecule. To clarify the mechanism of energy transduction, to quantify the migration of those ions seems to be indispensable.Conformational changes in H^+ pump were studied using intrinsic (tryptophan) and extrinsic (BIPM, FITC) fluorescence probes. Fluorescence intensity of Trp residues increases after formation of phosphoenzymes. Addition of K^+ to the phosphoenzyme reduced the fluorescence to the same level to that of HE_1. Fluorescence intensity of BIPM probe decreased with nearly the same rate constant of phosphorylation. Addition of K^+ to the phosphoenzyme slightly increased the BIPM fluorescence. These data clearly show that both fluorescence probes sense different enzyme states as already shown in Na^+ pump using various fluorescence probes.3. Na^+ pump was modified to label the fluorescence probe near ATP binding domain with pyridoxal phosphate in the presence of Na^+ or K^+. Na^+, K^+-ATPase activity and phosphorylation from ATP were inhibited while phosphorylation from acetyl phosphate remained. The sites of pyridoxal phosphate probe attached seemed to be Lys-664 in NaE_1 and both Lys-664 and Lys-469 in KE_2.4. Role of phospholipids in the conformational change in Na^+, K^+-ATPase were studied. The results showed that the change in the fluorescence intensity of BIPM probe at Cys-964 accompanying accumulation of E_2P is more susceptible than that of E_1P to the phospholipase A treatment.5. Cyclic AMP-dependent protein kinase was purified from pig kidney red outer medulla from which Na^+, K^+-ATPase has been purified. Na^+, K^+-ATPase was phosphorylated by the purified kinase. One mole of phosphate was detected in the one mole of alpha-chain sequence for the site of phosphorylation and the change in the Na^+, K^+-ATPase activity and the affinity for ligands after phosphorylation by the kinase are now in progress. Less