Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay.
Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay.
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通过简单的手动测定来表征肾 (Na K)-ATP 酶中的 Rb 闭塞。
DOI：
10.1016/0005-2736(87)90081-2
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
S. Karlish
中科院分区：
文献类型：
--
作者：
M. Shani;R. Goldschleger;S. Karlish
本文描述了一种简单的手工检测Rb+阻断肾(Na++ K+)- atp酶的特性。将酶加Rb+(86 Rb)混合物置于Dowex-50阳离子交换柱上，在0°C下测定Rb+的闭塞度，并用冰冷的蔗糖溶液将封闭的Rb+洗脱酶。酶-Rb+络合物在0°c时相当稳定。该方法可用于测量平衡结合条件下Rb+的遮挡和酶-Rb+络合物的缓慢解离速率。每个磷酸化位点Rb+的化学计量为2。Rb+饱和曲线为严格的双曲型，表明两个Rb+位点具有非常不同的亲和关系，一个在微摩尔范围内，一个在几十毫摩尔范围内。ATP将Rb+饱和度曲线向右移动（对照k0.5 100 - 200 μM；在100mm Tris-HCl培养基中，pH 7.0中，加上ATP， k0.5 0.8-1.4 mM），并降低了最大遮挡水平（对照约为0.96 μM）。4 nmol /毫克;加上ATP。3 nmol/mg蛋白质)。因此，正如预期的那样，ATP将e1 2Rb+- e2 (2Rb+) occ平衡向e1转移。在Tris-HCl介质中，浓度高达30mm的钠离子与kna = 1.86 mM的铷离子竞争。较高浓度（30-100 mM）的Na+具有额外的非竞争性拮抗作用。在室温下，Rb+在Rb+(20 mM)或Na （100 mM）存在下缓慢地从酶解离，k obs= 0.08 s−1。正如预期的那样，ATP极大地加速了解离，这种技术无法测量解离的速度。（Na++ K+）- atp酶在Na+培养基中被凝乳胰蛋白酶选择性水解，阻断Rb+。对于对照和蛋白水解(Na++ K+)- atp酶，Rb+饱和曲线相似，酶-Rb+复合物的解离速率相同。胰凝乳分裂似乎破坏了阳离子和ATP结合域之间的拮抗相互作用，而未磷酸化蛋白的e1 - e2构象转变可能仍然存在。
This paper describes properties of a simple manual assay for Rb+ occlusion on renal (Na++ K+)-ATPase. Rb+ occlusion is measured by applying the enzyme plus Rb+(86 Rb) mixture to a Dowex-50 cation exchange column at 0° C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0° C. This method is useful for measuring Rb+ occlusion under equilibrium binding conditions and slow rates of dissociation of the enzyme-Rb+ complex. The stoichiometry of Rb+ occluded per phosphorylation site is 2. Rb+ saturation curves are strictly hyperbolic, suggesting that the two Rb+ sites have very different affinities, one in the micromolar range and one in the tens of millimolar range. ATP shifts the Rb+ saturation curves to the right (control K 0.5 100–200 μM; plus ATP, K 0.5 0.8–1.4 mM, in a 100 mM Tris-HCl medium, pH 7.0) and reduces the maximal level occluded (control approx. 4 nmol/mg; plus ATP approx. 3 nmol/mg protein). Thus, as expected, ATP shifts the E 1 2Rb+-E 2 (2Rb+) occ equilibrium towards E 1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, K Na= 1.86 mM in the Tris-HCl medium. Na+ at higher concentrations (30–100 mM) has an added non-competitive antagonistic effect. At room temperature, Rb+ dissociates slowly from the enzyme, k obs= 0.08 s− 1, in the presence of either Rb+(20 mM) or Na,(100 mM). As expected, dissociation is greatly accelerated by ATP, the rate being to fast to be measured by this technique.(Na++ K+)-ATPase proteolyzed selectively by chymotrypsin in a Na+ medium, occludes Rb+. For control and proteolyzed (Na++ K+)-ATPase the Rb+ saturation curves are similar and the rates of dissociation of the enzyme-Rb+ complex are identical. The chymotryptic split appears to disrupt antagonistic interactions between cation and ATP binding domains, while the E 1-E 2 conformational transition of the unphosphorylated protein probably remains.