Sarvazyan N A, Modyanov N N, Askari A
Department of Pharmacology, Medical College of Ohio, Toledo 43699-0008, USA.
J Biol Chem. 1995 Nov 3;270(44):26528-32. doi: 10.1074/jbc.270.44.26528.
To identify interfaces of alpha- and beta-subunits of Na+/K(+)-ATPase, and contact points between different regions of the same alpha-subunit, purified kidney enzyme preparations whose alpha-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit alpha,alpha-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an alpha,beta-dimer, a dimer of N-terminal and C-terminal alpha fragments, and a trimer of beta and the two alpha fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of alpha, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of alpha (H1-H2). The other was a trimer of beta, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an alpha,beta-dimer and a dimer of beta and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of alpha, these findings indicate that (a) the single intramembrane helix of beta is in contact with portions of H8-H10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of alpha; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.
为了确定Na⁺/K⁺-ATP酶α亚基和β亚基的界面，以及同一α亚基不同区域之间的接触点，对经不同方式进行可控蛋白水解的纯化肾酶制剂，用洋地黄皂苷使其溶解以破坏亚基间α-α相互作用，并进行氧化交联。通过凝胶电泳、特异性抗体染色和N端分析鉴定出以下二硫键交联产物。1）在Arg438-Ala439处部分裂解的酶中，交联产物为α-β二聚体、N端和C端α片段的二聚体，以及β和两个α片段的三聚体。2）从含有22 kDa C端和几个较小α片段的充分消化的酶中获得了两种交联产物。一种是22 kDa C端肽与含有α的前两个跨膜螺旋（H1-H2）的11 kDa N端肽的二聚体。另一种是β、11 kDa和22 kDa肽的三聚体。3）在Leu266-Ala267处部分裂解的制剂的交联产物为α-β二聚体以及β与83 kDa C端片段的二聚体。假设α最可能的10跨膜模型，这些发现表明：（a）β的单个跨膜螺旋与α的H8-H10跨膜螺旋部分接触；（b）α的N端H1-H2和C端H8-H10片段之间有紧密接触；最可能相互作用的螺旋是H1、H10对和H2、H8对。
