单价阳离子与从猪肾中纯化的钠钾依赖型ATP酶的结合。I. 三个钠离子和两个钾离子或铷离子与该酶的同时结合。

Binding of monovalent cations to Na+,K+-dependent ATPase purified from porcine kidney. I. Simultaneous binding of three sodium and two potassium or rubidium ions to the enzyme.

作者信息

Yamaguchi M, Tonomura Y

出版信息

J Biochem. 1980 Nov;88(5):1365-75. doi: 10.1093/oxfordjournals.jbchem.a133105.

DOI:10.1093/oxfordjournals.jbchem.a133105

Abstract

We previously measured the amounts of Na+ and K+ ions bound to the Na+,K+-dependent ATPase [EC 3.6.1.3] purified from porcine kidney by a modified membrane filtration method [(1979) J. Biochem. 86, 509--523]. In this study, we improved the method for measuring the amount of the active site and measured the amount of Rb+ ions (a K+ congener) bound to the ATPase as well as those of Na+ and K+ ions to get more accurate information on the K+- and Na+-binding sites. The following results were obtained. Two kinds of cation-binding sites were found to exist on the ATPase molecule. One was the Na+-binding sites (3 mol per mol of active site). Na+ ions were bound to the sites cooperatively (Hill coefficient, 2.5--3), and the apparent dissociation constant was 0.20--0.32 mM. Three moles of Na+ ions bound to the sites was displaced by 1 mol of K+ ions bound to the ATPase (phi K, 24 microM). The other was the K+-binding sites (2 mol per mol of active site). Two moles of K+, Rb+, or Na+ ions was bound to the sites cooperatively (Hill coefficient, 1.5--2), and their apparent dissociation constants were 0.044, 0.024, and 2.2 mM, respectively. We measured the amounts of Na+ and Rb+ ions bound to the ATPase in the presence of 0.8 mM NaCl and 0.13 mM RbCl, and obtained unequivocal evidence for the simultaneous binding of 3 mol of Na+ ions and 2 mol of Rb+ ions per mol of active site of the ATPase.

摘要

我们之前通过改良的膜过滤法[(1979) J. Biochem. 86, 509 - 523]测量了从猪肾中纯化得到的Na⁺,K⁺-依赖型ATP酶[EC 3.6.1.3]结合的Na⁺和K⁺离子量。在本研究中，我们改进了测量活性位点数量的方法，并测量了结合到该ATP酶上的Rb⁺离子（一种K⁺同类物）以及Na⁺和K⁺离子的量，以获取关于K⁺和Na⁺结合位点更准确的信息。得到了以下结果。发现ATP酶分子上存在两种阳离子结合位点。一种是Na⁺结合位点（每摩尔活性位点3摩尔）。Na⁺离子协同结合到这些位点上（希尔系数，2.5 - 3），表观解离常数为0.20 - 0.32 mM。结合到该ATP酶上的1摩尔K⁺离子可取代结合到这些位点上的3摩尔Na⁺离子（φK，24 μM）。另一种是K⁺结合位点（每摩尔活性位点2摩尔）。2摩尔的K⁺、Rb⁺或Na⁺离子协同结合到这些位点上（希尔系数，1.5 - 2），它们的表观解离常数分别为0.044、0.024和2.2 mM。我们在0.8 mM NaCl和0.13 mM RbCl存在的情况下测量了结合到该ATP酶上的Na⁺和Rb⁺离子量，得到了明确的证据，表明每摩尔ATP酶活性位点同时结合3摩尔Na⁺离子和2摩尔Rb⁺离子。