Cys<SUP>577</SUP> is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase α-subunit

作     者：Gatto, C Thornewell, SJ Holden, JP Kaplan, JH 

作者机构：Oregon Hlth & Sci Univ Dept Biochem & Mol Biol Portland OR 97201 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第35期

页      面：24995-25003页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：NHLBI NIH HHS [HL09972] Funding Source: Medline NIGMS NIH HHS [GM39500] Funding Source: Medline 

主　　题：腺苷三磷酸/代谢 苯胺基萘磺酸类/药理学 结合部位 阳离子/药理学 色谱法 高压液相 半胱氨酸/化学 电泳 聚丙烯酰氨凝胶 酶抑制剂/药理学 动力学 肽碎片/化学 蛋白质结合 序列分析 钠钾交换ATP酶/化学 胰蛋白酶 

摘      要：2-[4 -Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K+ (1 mM);the protective effect K+ is reversed at higher concentrations, This biphasic effect was also observed with K+ congeners, In contrast, Na+ ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography, ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of similar to 5 kDa, N-terminal. amino acid sequencing identified the peptide (V(545)LGFCH...), This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.