Tal D M, Capasso J M, Munson K, Karlish S J
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Biochemistry. 2001 Oct 23;40(42):12505-14. doi: 10.1021/bi011167n.
This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.
本文描述了一种新颖的方法，可在抗坏血酸盐和H₂O₂存在的情况下，由Cu²⁺离子和一种疏水性菲咯啉4,7 - 二苯基 - 1,10 - 菲咯啉（DPP）介导对Na⁺,K⁺ - ATP酶进行特异性氧化裂解。裂解产生α亚基的两个主要片段，表观分子量分别为96.5 kDa和76 kDa，其N端分别靠近跨膜片段M1和M3的细胞质入口。动力学表明，两种裂解均由单一的Cu²⁺ - DPP复合物介导。我们推断M3和M1在细胞质表面附近彼此靠近。稳定不同E₁和E₂构象的配体对96.5 kDa和76 kDa片段的产量没有显著影响。在E₂(K)和E₂P构象中，还观察到一个较小的5.5 kDa片段，其N端位于M10。96.5 kDa和76 kDa片段与先前描述的由Fe²⁺催化裂解产生的靠近M3和M1的两个片段无法区分[Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213 - 16221]，而使用Cu²⁺ - DPP复合物未观察到细胞质P和A结构域中其他由Fe²⁺催化产生的裂解片段。这些发现为两个独立的Fe²⁺位点的概念提供了实验支持。一个同源模型，将跨膜片段和连接环内的Na⁺,K⁺ - ATP酶残基替换到Ca²⁺ - ATP酶的晶体结构中，显示了细胞质表面附近M3中的HFIH序列与M1中的EVWK序列之间的接近性。因此，该模型有力地支持了基于Cu²⁺ - DPP复合物（或位点2处的Fe²⁺）介导的裂解得出的结论。作为一个推论，这些裂解为Na⁺,K⁺ - ATP酶和Ca²⁺ - ATP酶的M1和M3的相似堆积提供了证据。
