INTERACTIONS OF PHOSPHORYLATION AND DIMERIZING DOMAINS OF THE ALPHA-SUBUNITS OF NA+ K+-ATPASE/

Chemical cross-linking studies are among a number of experimental appr oaches that have suggested the functional significance of higher assoc iation states of alpha,beta-protomers of Na+/K+-ATPase. Formation of t he phosphointermediate of the enzyme on Asp(369) Of the alpha-subunit is known to induce oxidative cross-linking of the alpha-subunits catal yzed by Cu2+-phenanthroline. To localize the phosphorylation-induced a lpha,alpha-interface, we cleaved alpha at Arg(438)-Ala(439) by control led proteolysis and exposed the partially cleaved enzyme to the cross- linking reagent. In addition to the alpha,alpha-dimer, two other phosp horylation-induced cross-linked products were obtained, Using gel elec trophoretic resolution of the cross-linked P-32-labeled enzyme, N-term inal analyses of the products, and their reactivities with sequence-sp ecific antibodies, the two products were identified as a homodimer of the C-terminal 64-kDa fragment of alpha and a heterodimer of alpha and the 64-kDa peptide. The latter dimer was also obtained when the cross -linked alpha,alpha-dimer was formed first and then subjected to prote olysis, The findings localize the dimerizing domain to the C-terminal side of Ala(439) and indicate that intersubunit proximities of dimeriz ing domains are regulated by phosphorylation-dephosphorylation of Asp( 369) during the reaction cycle of the enzyme.