Capasso J M, Hoving S, Tal D M, Goldshleger R, Karlish S J
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1992 Jan 15;267(2):1150-8.
This paper extends our recent report that renal Na+,K(+)-ATPase is digested by trypsin in the absence of Ca2+ and presence of Rb+ ions to a stable 19-kDa fragment and smaller membrane-embedded fragments of the alpha chain and essentially intact beta chain. These are referred to as "19-kDa membranes." Occlusion of both Rb+ (K+) or Na+ ions is preserved, but ATP-dependent functions are lost (Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570). We now show that extensive digestion with nonselective fungal proteases (Pronase and proteinase K) alone, in combination, or after tryptic digestion can remove up to 70% of membrane protein without destroying Rb+ occlusion. In the most heavily digested membranes, the 19-kDa fragment or a slightly shorter 18.5-kDa fragment and smaller fragments of the alpha chain remain, whereas the beta chain is largely digested, leaving smaller membrane-embedded fragments (13-15 kDa). For either trypsin or Pronase digestion, preservation of Rb+ occlusion and the specific fragmentation pattern is observed only in the absence of divalent metal ions (Mg2+ or Ca2+) and presence of either Rb+ or Na+ or congener ions. Tryptic digestion at pH 7.0 can split the beta chain into two fragments of approximately 50 and 16 kDa joined by an S-S bridge. The 16-kDa fragment is protected against further digestion by the presence of Rb+ ions, but probably is not directly involved in occluding cations. Tryptic 19-kDa membranes show a clear and reproducible fragmentation pattern in which all predicted membrane segments are identifiable. Families of fragments from 19-kDa membranes, including seven peptides of 7.6-11.7 kDa, have been separated by size-exclusion high performance liquid chromatography, concentrated, and resolved on 16.5% Tricine gels. N-terminal sequences of the different fragments have been determined after transfer to polyvinylidene difluoride paper. The most interesting findings are as follows. (a) Whereas the 19-kDa tryptic fragment begins at Asn831 as reported previously, the 18.5-kDa Pronase fragment begins at Thr834. (b) Fragments in tryptic 19-kDa membranes of 7.6-11.7 kDa begin at Asp68, Ile263, and Gln737, respectively. These include all putative transmembrane segments other than those in the 19-kDa fragment. (c) A Pronase fragment of 7.8 kDa begins at Thr834, i.e. apparently the 19-kDa fragment has been partially cut, without loss of Rb+ occlusion. (d) Tryptic 16- and approximately 50-kDa fragments of the beta chain begin at Ala5 and Gly143, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
本文扩展了我们最近的报道，即在没有Ca2+且存在Rb+离子的情况下，肾Na+,K(+)-ATP酶被胰蛋白酶消化成一个稳定的19 kDa片段以及α链的较小膜嵌入片段和基本完整的β链。这些被称为“19 kDa膜”。Rb+（K+）或Na+离子的封闭得以保留，但ATP依赖性功能丧失（卡里什，S.J.D.，戈德施莱格，R.，和斯坦，W.D.（1990年）美国国家科学院院刊87，4566 - 4570）。我们现在表明，单独使用非选择性真菌蛋白酶（链霉蛋白酶和蛋白酶K）、联合使用或在胰蛋白酶消化后进行广泛消化，可去除高达70%的膜蛋白而不破坏Rb+封闭。在消化最严重的膜中，19 kDa片段或稍短的18.5 kDa片段以及α链的较小片段保留下来，而β链大部分被消化，留下较小的膜嵌入片段（13 - 15 kDa）。对于胰蛋白酶或链霉蛋白酶消化，只有在没有二价金属离子（Mg2+或Ca2+）且存在Rb+或Na+或同类离子的情况下，才会观察到Rb+封闭的保留和特定的片段化模式。在pH 7.0下进行胰蛋白酶消化可将β链分成两个分别约为50 kDa和16 kDa的片段，通过一个S - S桥连接。16 kDa片段因Rb+离子的存在而受到保护不被进一步消化，但可能不直接参与阳离子封闭。胰蛋白酶处理的19 kDa膜呈现出清晰且可重复的片段化模式，其中所有预测的膜段都可识别。来自19 kDa膜的片段家族，包括7个7.6 - 11.7 kDa的肽段，已通过尺寸排阻高效液相色谱分离、浓缩，并在16.5%的三羟甲基氨基甲烷凝胶上进行分离。不同片段的N端序列在转移到聚偏二氟乙烯纸上后已被确定。最有趣的发现如下。（a）如先前报道，19 kDa胰蛋白酶片段始于Asn831，而18.5 kDa链霉蛋白酶片段始于Thr834。（b）胰蛋白酶处理的19 kDa膜中7.6 - 11.7 kDa的片段分别始于Asp68、Ile263和Gln737。这些包括19 kDa片段以外的所有假定跨膜段。（c）一个7.8 kDa的链霉蛋白酶片段始于Thr834，即显然19 kDa片段已被部分切割，但Rb+封闭未丧失。（d）β链的胰蛋白酶处理的16 kDa和约50 kDa片段分别始于Ala5和Gly143。（摘要截断于400字）
