Ganjeizadeh M, Zolotarjova N, Huang W H, Askari A
Department of Pharmacology, Medical College of Ohio, Toledo 43699-0008, USA.
J Biol Chem. 1995 Jun 30;270(26):15707-10. doi: 10.1074/jbc.270.26.15707.
Chemical cross-linking studies are among a number of experimental approaches that have suggested the functional significance of higher association states of alpha,beta-protomers of Na+/K(+)-ATPase. Formation of the phosphointermediate of the enzyme on Asp369 of the alpha-subunit is known to induce oxidative cross-linking of the alpha-subunits catalyzed by Cu(2+)-phenanthroline. To localize the phosphorylation-induced alpha,alpha-interface, we cleaved alpha at Arg438-Ala439 by controlled proteolysis and exposed the partially cleaved enzyme to the cross-linking reagent. In addition to the alpha,alpha-dimer, two other phosphorylation-induced cross-linked products were obtained. Using gel electrophoretic resolution of the cross-linked 32P-labeled enzyme, N-terminal analyses of the products, and their reactivities with sequence-specific antibodies, the two products were identified as a homodimer of the C-terminal 64-kDa fragment of alpha and a heterodimer of alpha and the 64-kDa peptide. The latter dimer was also obtained when the cross-linked alpha,alpha-dimer was formed first and then subjected to proteolysis. The findings localize the dimerizing domain to the C-terminal side of Ala439 and indicate that intersubunit proximities of dimerizing domains are regulated by phosphorylation-dephosphorylation of Asp369 during the reaction cycle of the enzyme.
化学交联研究是众多实验方法之一，这些方法表明了Na⁺/K⁺-ATP酶α、β原聚体更高缔合状态的功能意义。已知在α亚基的Asp369上形成酶的磷酸中间体可诱导由Cu(2+)-菲咯啉催化的α亚基的氧化交联。为了定位磷酸化诱导的α,α界面，我们通过可控蛋白酶解在Arg438-Ala439处切割α，并将部分切割的酶暴露于交联剂。除了α,α二聚体，还获得了另外两种磷酸化诱导的交联产物。通过对交联的³²P标记酶进行凝胶电泳分离、对产物进行N端分析以及它们与序列特异性抗体的反应性，这两种产物被鉴定为α的C端64-kDa片段的同二聚体以及α与64-kDa肽的异二聚体。当先形成交联的α,α二聚体然后进行蛋白酶解时，也获得了后一种二聚体。这些发现将二聚化结构域定位到Ala439的C端一侧，并表明在酶的反应循环中，二聚化结构域的亚基间接近度受Asp369的磷酸化-去磷酸化调节。
