Intersubunit and intrasubunit contact regions of Na+/K+-ATPase revealed by controlled proteolysis and chemical cross-linking

Abstract

To identify interfaces of α- and β-subunits of Na+/K+-ATPase, and contact points between different regions of the same α-subunit, purified kidney enzyme preparations whose α-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit α,α-interactions, and oxidatively cross-linked. The following disulfide crosslinked 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 α,β-dimer, a dimer of N-terminal and C-terminal α fragments, and a trimer of β and the two α fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of α, 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 α (H1-H2). The other was a trimer of β, the 11- kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an α,β-dimer and a dimer of β and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of α, these findings indicate that (α) the single intramembrane helix of β is in contact with portions of H8-H10 intramembrane helices of α; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of α; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.