Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.
Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.
复制标题
Na ,K( )-ATPase α 亚基的 M5-M6 发夹的膜配置是配体依赖性的。
DOI：
10.1073/pnas.92.17.7936
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发表时间：
1995
影响因子：
11.1
通讯作者：
Kaplan,JH
中科院分区：
文献类型：
--
作者：
Lutsenko,S;Anderko,R;Kaplan,JH
通过胰蛋白酶对Na+，K（+）-ATP酶（EC 3.6.1.37）进行广泛的蛋白水解消化产生了一种制剂，其中α亚基的大部分膜外部分已被消化掉，而β亚基基本上保持完整。在37 ℃孵育几分钟后，Na+，K（+）-ATP酶α亚基的片段Gln-737-Arg-829（包括推定的跨膜发夹M5-M6）容易、选择性和不可逆地从胰蛋白酶后膜制备物中释放。一旦从膜释放，片段聚集但保持水溶性。K+或Rb+的封闭特异性阻止Gln-737-Arg-829片段释放到上清液中。用半胱氨酸定向试剂标记胰蛋白酶后膜制备物显示，Cys-802（被认为位于M6片段内）被保护免受Rb+的修饰，而该片段在膜中，但在释放时可以容易地被修饰。阳离子封闭明显改变了M5-M6片段在膜中的折叠和/或处置，其方式在片段迁移到水相时不会发生。M5-M6发夹在膜中的配体依赖性分布沿着最近的标记研究表明，该片段在Na+，K（+）-ATP酶的阳离子泵送中起关键作用。
Extensive proteolytic digestion of Na+,K(+)-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the alpha subunit have been digested away and the beta subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na+,K(+)-ATPase alpha subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37 degrees C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K+ or Rb+ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine-directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb+ while this fragment is in the membrane but can be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the M5-M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na+,K(+)-ATPase.