SPECIFIC CROSS-LINKS BETWEEN FRAGMENTS OF PROTEOLYZED NA,K-ATPASE INDUCED BY O-PHTHALALDEHYDE

We have used o-phthalaldehyde (OPA) to cross-link adjacent fragments o f ''19 kDa membranes'', a tryptic preparation of Na,K-ATPase lacking t he ATP site but retaining cation occlusion sites. Treatment with OPA o f ''19 kDa membranes'' or detergent-solubilized membranes containing o ccluded Rb ions [Or, E., Goldshleger, R,, Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864] yielded crosslinked products of 25 and 31 kDa. Both species contained the 19 kDa fragment of the ct subunit (transmembrane segments M7-M10). In addition, the 25 kDa prod uct contained the fragment including M5-M6, while the 31 kDa product c ontained a 16 kDa fragment of the beta subunit. Cross-linking was unaf fected by the absence or presence of ligands (Na, Rb, or Mg and ouabai n). Cross-linking was largely abolished in thermally inactivated ''19 kDa membranes''. When proteolytic digestion of the 25 and 31 kDa produ cts was combined with antibody binding, PKA-dependent phosphorylation, and sequencing of fragments, approximate positions of the cross-links were established. In the 25 kDa product, the crosslink was located wi thin the short cytoplasmic segment Asn831-Arg841 of the 19 kDa fragmen t preceding M7 and within Ala749-Ala770 preceding M5. Thus, M7 and M5 are likely to be in close proximity. In the 31 kDa product, the cross- link was located in the extracellular loop of the alpha subunit betwee n M7 and M8, close to residues which are known to interact with the be ta subunit. Functional implications of the interactions between the fr agments of the alpha (M5-M6 and M7-M10) and beta subunits are discusse d.