Abstract
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.
Highlights
Naϩ/Kϩ-ATPase is the intrinsic enzyme of the plasma membrane that carries out the coupled active transport of Naϩ and Kϩ in most eucaryotic cells
1) In the enzyme that was partially cleaved at Arg438Ala439, the cross-linked products were an ␣,-dimer, a dimer of N-terminal and C-terminal ␣ fragments, and a trimer of  and the two ␣ fragments
More recent studies have indicated the role of the -subunit in the regulation of Kϩ binding to the enzyme (10 –12), and have provided further evidence for the involvement of both subunits in the enzyme’s conformational transitions [13, 14]
Summary
Purified membrane-bound Naϩ/Kϩ-ATPase of canine kidney medulla, with specific activity in the range of 1000 –1600 mol of ATP hydrolyzed/mg of protein/h, was prepared and assayed as described before [26]. Such preparations were subjected to controlled proteolysis by the following well established procedures. Two additional polyclonal antibodies were obtained by conventional immunization of rabbits using denatured ␣- and -subunits of canine kidney Naϩ/Kϩ-ATPase as antigens. These were prepared by resolving sufficient quantities of the purified enzyme on preparative SDS-polyacrylamide gels, and electroelution of the separated subunits
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