title: Interactions of phosphorylation and dimerizing domains of the alpha-subunits of Na+/K(+)-ATPase.
abstract: Chemical cross-linking studies are among a number of experimental approaches that have suggested the functional significance of higher association states of alpha,beta-protomers of Na+/K(+)-ATPase. Formation of the phosphointermediate of the enzyme on Asp369 of the alpha-subunit is known to induce oxidative cross-linking of the alpha-subunits catalyzed by Cu(2+)-phenanthroline. To localize the phosphorylation-induced alpha,alpha-interface, we cleaved alpha at Arg438-Ala439 by controlled proteolysis and exposed the partially cleaved enzyme to the cross-linking reagent. In addition to the alpha,alpha-dimer, two other phosphorylation-induced cross-linked products were obtained. Using gel electrophoretic resolution of the cross-linked 32P-labeled enzyme, N-terminal analyses of the products, and their reactivities with sequence-specific 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 proteolysis. The findings localize the dimerizing domain to the C-terminal side of Ala439 and indicate that intersubunit proximities of dimerizing domains are regulated by phosphorylation-dephosphorylation of Asp369 during the reaction cycle of the enzyme.
authors.affiliation: Department of Pharmacology, Medical College of Ohio, Toledo 43699-0008, USA.
authors.forename: M
authors.initials: M
authors.lastname: Ganjeizadeh
authors.forename: N
authors.initials: N
authors.lastname: Zolotarjova
authors.forename: W H
authors.initials: WH
authors.lastname: Huang
authors.forename: A
authors.initials: A
authors.lastname: Askari
mesh_terms: D000818:Animals; D004285:Dogs; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase