Tadini-Buoninsegni Francesco, Nassi Paolo, Nediani Chiara, Dolfi Andrea, Guidelli Rolando
Department of Chemistry, Florence University, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy.
Biochim Biophys Acta. 2003 Apr 1;1611(1-2):70-80. doi: 10.1016/s0005-2736(02)00722-8.
Charge translocation by Na(+),K(+)-ATPase was investigated by adsorbing membrane fragments containing Na(+),K(+)-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na(+),K(+)-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na(+) and K(+) ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme alpha-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na(+),K(+)-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na(+) but not of K(+), the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na(+),K(+)-ATPase.
通过将含有猪肾钠钾ATP酶的膜片段吸附在固体支撑膜（SSM）上来研究钠钾ATP酶的电荷转运。吸附后，通过在SSM表面进行ATP浓度阶跃来激活离子泵，并在恒电位条件下测量钠钾ATP酶产生的电容电流瞬变。为了研究离子泵在多次周转条件下的行为，在存在钠离子和钾离子的情况下进行了ATP浓度阶跃实验。在存在胰凝乳蛋白酶的情况下也记录了由ATP浓度阶跃引起的电流瞬变。通过在存在和不存在酰基磷酸酶（AcP）的情况下进行ATP浓度阶跃，研究了一种胞质酶酰基磷酸酶（AcP）的影响，该酶可能通过水解其酰基磷酸化中间体来影响钠钾ATP酶的功能。在存在钠离子但不存在钾离子的情况下，添加AcP会导致因ATP浓度阶跃而转移的电荷在pH值从6到7的范围内减少约50%，在pH值为8时增加约20%。相反，在不存在AcP的情况下，未观察到pH值对转移电荷有明显影响。上述行为表明质子参与了钠钾ATP酶酰基磷酸化中间体的AcP催化去磷酸化过程。
