Shainskaya A, Karlish S J
Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
J Biol Chem. 1996 Apr 26;271(17):10309-16. doi: 10.1074/jbc.271.17.10309.
This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the beta subunit of Na/K-ATPase leads to changes in the kinetics of occlusion of Rb+ ions. The experiments utilize extensively trypsinized Na/K-ATPase, "19-kDa membranes," which lack cytoplasmic loops of the alpha subunit, whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three fragments of 8.1-11.7 kDa) containing transmembrane segments and extracellular loops are intact. The beta subunit is partially split into NH2- and COOH-terminal fragments of 16 and approximately 50 kDa, respectively. Cation occlusion and ouabain binding are preserved. The 19-kDa membranes were incubated, at 37 degrees C, with a selection of proteases, in the presence of Rb+ ions. In these conditions, only alpha-chymotrypsin destroyed the ability to occlude Rb+ ions. This process was associated with truncation of the 16-kDa fragment of the beta subunit in two stages. In the first stage, chymotrypsin removed 10 residues from the 16-kDa fragment to form a 15-kDa fragment (NH2-terminal Ile15) and 4 or 6 residues from the NH2 terminus of the alpha subunit fragment beginning at Asp68. In these membranes Rb+ occlusion was still intact at 37 degrees C. Strikingly, however, deocclusion of two Rb+ ions, which is characteristically biphasic in 19-kDa membranes, displayed deocclusion kinetic with mainly one fast phase. These membranes also showed a much lower affinity for Rb+ ions compared with 19-kDa membranes; and, consistent with the lower Rb+ affinity, Rb+ ions, at nonsaturating concentrations, protected less well against thermal inactivation of Rb+ occlusion. In the second stage, the 15-kDa fragment was truncated further to a 14-kDa fragment (NH2-terminal Leu24), followed by thermal destabilization of Rb+ occlusion even at high concentrations of Rb+ ions. Eventually, the thermally inactivated complex of fragments of alpha and beta subunits was digested to the limit peptides. The results suggest that the cytoplasmic domain of the beta subunit interacts with that of the alpha subunit, possibly with residues leading into the first transmembrane segment, and controls access of Rb+ ions into or out of the occlusion sites.
本文表明，对钠钾-ATP酶β亚基的胞质结构域进行特定的胰凝乳蛋白酶消化会导致铷离子封闭动力学发生变化。实验使用了经过广泛胰蛋白酶处理的钠钾-ATP酶“19-kDa膜”，其缺乏α亚基的胞质环，而包含跨膜片段和细胞外环的膜嵌入片段（一个COOH末端19 kDa和三个8.1-11.7 kDa的片段）是完整的。β亚基被部分切割成分别为16 kDa和大约50 kDa的NH2-和COOH-末端片段。阳离子封闭和哇巴因结合得以保留。将19-kDa膜在37℃下于铷离子存在的情况下与多种蛋白酶一起孵育。在这些条件下，只有α-胰凝乳蛋白酶破坏了封闭铷离子的能力。这个过程与β亚基16-kDa片段的截断分两个阶段相关。在第一阶段，胰凝乳蛋白酶从16-kDa片段去除10个残基以形成一个15-kDa片段（NH2-末端Ile15），并从α亚基片段的NH2末端（从Asp68开始）去除4或6个残基。在这些膜中，铷离子封闭在37℃时仍然完整。然而，引人注目的是，两个铷离子（在19-kDa膜中其解封闭具有典型的双相性）的解封闭呈现出主要为一个快速相的解封闭动力学。这些膜与19-kDa膜相比，对铷离子的亲和力也低得多；并且，与较低的铷离子亲和力一致，在非饱和浓度下，铷离子对铷离子封闭的热失活的保护作用也较差。在第二阶段，15-kDa片段进一步截断为一个14-kDa片段（NH2-末端Leu24），随后即使在高浓度铷离子存在的情况下，铷离子封闭也发生热不稳定。最终，α和β亚基片段的热失活复合物被消化成极限肽段。结果表明，β亚基的胞质结构域与α亚基的胞质结构域相互作用，可能与通向第一个跨膜片段的残基相互作用，并控制铷离子进出封闭位点的通道。
