用一种简单的手工测定法对肾脏（钠 + 钾）-ATP 酶中的铷离子阻断进行表征。

Rb+ occlusion in renal (Na+ + K+)-ATPase characterized with a simple manual assay.

作者信息

Shani M, Goldschleger R, Karlish S J

机构信息

Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.

出版信息

Biochim Biophys Acta. 1987 Nov 2;904(1):13-21. doi: 10.1016/0005-2736(87)90081-2.

DOI:10.1016/0005-2736(87)90081-2

PMID:2822111

Abstract

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+ (86Rb) mixture to a Dowex-50 cation exchange column at 0 degree C, and eluting the enzyme with occluded Rb+ using an ice-cold sucrose solution. The enzyme-Rb+ complex is quite stable at 0 degree 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 K0.5 100-200 microM; plus ATP, K0.5 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+-E2(2Rb+)occ equilibrium towards E1. Sodium ions at concentrations of up to 30 mM compete with the rubidium ions, KNa = 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, kobs = 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 E1-E2 conformational transition of the unphosphorylated protein probably remains.

摘要

本文描述了一种用于检测肾（Na⁺ + K⁺）-ATP酶上Rb⁺封闭作用的简单手工测定法的特性。Rb⁺封闭作用的测定方法是，在0℃下将酶与Rb⁺（⁸⁶Rb）混合物加到Dowex - 50阳离子交换柱上，并用冰冷的蔗糖溶液洗脱被封闭Rb⁺的酶。酶 - Rb⁺复合物在0℃时相当稳定。该方法可用于在平衡结合条件下以及酶 - Rb⁺复合物解离速率较慢的情况下测定Rb⁺封闭作用。每个磷酸化位点所封闭的Rb⁺的化学计量比为2。Rb⁺饱和曲线严格呈双曲线，这表明两个Rb⁺位点具有非常不同的亲和力，一个在微摩尔范围内，另一个在数十毫摩尔范围内。ATP使Rb⁺饱和曲线右移（在100 mM Tris - HCl介质，pH 7.0中，对照K₀.₅为100 - 200 μM；加ATP时，K₀.₅为0.8 - 1.4 mM），并降低最大封闭水平（对照约为4 nmol/mg；加ATP时约为3 nmol/mg蛋白质）。因此，正如预期的那样，ATP使E(1)2Rb⁺ - E2(2Rb⁺)封闭平衡向E1方向移动。浓度高达30 mM的钠离子与铷离子竞争，在Tris - HCl介质中KNa = 1.86 mM。更高浓度（30 - 100 mM）的Na⁺具有额外的非竞争性拮抗作用。在室温下，在存在Rb⁺（20 mM）或Na⁺（100 mM）的情况下，Rb⁺从酶上缓慢解离，观测到的解离速率常数kobs = 0.08 s⁻¹。正如预期的那样，ATP极大地加速了解离，其速率太快以至于无法用该技术测量。在Na⁺介质中被胰凝乳蛋白酶选择性水解的（Na⁺ + K⁺）-ATP酶会封闭Rb⁺。对于对照和经蛋白酶水解的（Na⁺ + K⁺）-ATP酶，Rb⁺饱和曲线相似，酶 - Rb⁺复合物的解离速率相同。胰凝乳蛋白酶的切割似乎破坏了阳离子与ATP结合结构域之间的拮抗相互作用，而未磷酸化蛋白质的E1 - E2构象转变可能仍然存在。