Goldshleger R, Bar Shimon M, Or E, Karlish S J
Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Acta Physiol Scand Suppl. 1998 Aug;643:89-97.
This paper describes a novel technique for specific cleavage of renal Na/K-ATPase, based on bound transition metal ions. The approach might have application to other P-type pumps or membrane proteins. In one type of experiment, specific cleavages of the alpha subunit have been observed following incubation with ascorbate plus H2O2. Five fragments with intact C-terminals and complementary fragments with intact N-terminals are detectable. The beta subunit is not cleaved. Cleavages depend on the presence of contaminant or added submicromolar concentrations of Fe2+ ions. The results suggest that Fe2+ (or Fe3+) binds with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. The rate of cleavage is greatly affected by the conformational state of the protein, E1Na or E2(Rb), respectively. The findings provide information on spatial organization of the protein and suggest that the highly conserved regions of the alpha subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations, but move apart in the E1 or E1Na conformations. In a second application of this technique, added Cu2+ ions at micromolar concentrations, have been shown to catalyse specific cleavages of both alpha and beta subunits at the extracellular surface. The experiments provide evidence for trans-membrane topology and proximity between trans-membrane segments M5-M10 within the alpha subunit and for interacting segments of alpha and beta subunits. We discuss the implications of metal-catalysed cleavages for spatial organisation of transmembrane helices of the protein.
本文描述了一种基于结合的过渡金属离子特异性切割肾脏钠钾ATP酶的新技术。该方法可能适用于其他P型泵或膜蛋白。在一类实验中，观察到在与抗坏血酸加过氧化氢孵育后，α亚基发生了特异性切割。可检测到五个具有完整C端的片段和具有完整N端的互补片段。β亚基未被切割。切割取决于污染物的存在或添加的亚微摩尔浓度的Fe2+离子。结果表明，Fe2+（或Fe3+）在细胞质表面以高亲和力结合，并催化靠近Fe2+（或Fe3+）离子的肽键切割。切割速率分别受到蛋白质E1Na或E2(Rb)构象状态的极大影响。这些发现提供了关于蛋白质空间组织的信息，并表明α亚基在小和大细胞质环内的高度保守区域在E2或E2(Rb)构象中相互作用，但在E1或E1Na构象中分开。在该技术的第二个应用中，已证明添加微摩尔浓度的Cu2+离子可催化α和β亚基在细胞外表面的特异性切割。这些实验为α亚基内跨膜片段M5-M10之间的跨膜拓扑结构和接近程度以及α和β亚基的相互作用片段提供了证据。我们讨论了金属催化切割对该蛋白质跨膜螺旋空间组织的影响。
