Sarvazyan N A, Ivanov A, Modyanov N N, Askari A
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008, USA.
J Biol Chem. 1997 Mar 21;272(12):7855-8. doi: 10.1074/jbc.272.12.7855.
An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha-subunit that contain all 10 transmembrane alpha domains, and the beta-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha-fragment containing the H1-H2 helices and a 22-kDa alpha-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha-fragments and that fraction of beta whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H1-H2 and H7-H10 helices of alpha is altered by detergent solubilization and by structural changes in the extracellular domain of beta, and (c) the three-dimensional packing of the interacting transmembrane helices of alpha and beta are regulated by the specific ligands of the enzyme.
一种经广泛胰蛋白酶消化的钠钾ATP酶，仍保留结合钠、钾和哇巴因的能力，它由包含所有10个跨膜α结构域的α亚基的四个片段以及β亚基组成，其中一部分β亚基在精氨酸142 - 甘氨酸143处被切割。在先前的研究中，我们用去污剂溶解该制剂，并通过化学交联确定了亚基几个跨膜螺旋的相对位置。为了确定这些检测到的螺旋 - 螺旋接近度是否代表溶解前双层膜中存在的接近度，我们现在对相同消化酶的膜结合制剂进行了类似研究。在铜 - 菲咯啉催化的氧化巯基交联后，通过它们的迁移率和N端分析鉴定出两种主要产物。一种是包含H1 - H2螺旋的11 kDaα片段和包含H7 - H10螺旋的22 kDaα片段的二聚体。这种二聚体似乎与在溶解制剂中获得的相同。另一种产物是上述两个α片段和β亚基中细胞外结构域在精氨酸142 - 甘氨酸143处被切割的部分的三聚体。该产物与溶解制剂中的类似产物不同，后者包含未进行细胞外切割的主要部分β亚基。膜制剂的交联反应，但不是溶解制剂的交联反应，被钠、钾和哇巴因特异性抑制。这些发现表明：（a）α亚基的H1 - H2跨膜螺旋在溶解状态和膜结合状态下均与其一些H7 - H10螺旋相邻；（b）β亚基单个跨膜螺旋的残基与α亚基相互作用的H1 - H2和H7 - H10螺旋的排列因去污剂溶解以及β亚基细胞外结构域的结构变化而改变；（c）α亚基和β亚基相互作用的跨膜螺旋的三维堆积受该酶的特异性配体调节。
