INTERSUBUNIT AND INTRASUBUNIT CONTACT REGIONS OF NA+/K+-ATPASE REVEALED BY CONTROLLED PROTEOLYSIS AND CHEMICAL CROSS-LINKING

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 Arg(438)-Ala(439), the cross-linked products were an alpha.beta-dimer, a dimer of N-terminal and C-terminal or 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 or, two crosslinked 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 (H-1-H-2). 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 Leu(266)-Ala(267) were an alpha,beta-dimer and a dimer of beta and the 83-bDa 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 H-8-H-10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H-2 and C-terminal H-8-H-10 segments of alpha; with the most probable interacting helices being the H-1,H-10-pair and the H-2,H-8-pair.