title: Autoregulation of the phosphointermediate of Na+/K(+)-ATPase by the amino-terminal domain of the alpha-subunit.
abstract: Chymotryptic cleavage of the alpha-subunit of the canine kidney Na+/K(+)-ATPase in the presence of Na+ abolishes ATPase activity and yields an 83 kDa peptide from Ala 267 to the COOH-terminus. To test the proposal that E1 to E2 conformational transition is blocked in this modified enzyme, we have made a detailed comparison of its phosphorylation with that of the native enzyme by ATP. While phosphorylation of alpha is dependent on Na+ and prevented by K+, that of the 83 kDa peptide is modestly stimulated by Na+; and only this stimulation, but not the Na(+)-independent phosphorylation is inhibited by K+. Ouabain, which inhibits alpha-phosphorylation by ATP, activates Na(+)-independent phosphorylation of the 83 kDa peptide by ATP, and inhibits the Na(+)-stimulation of this process. While there is a ouabain-stimulated phosphorylation of alpha by Pi, the 83 kDa peptide is not phosphorylated by Pi with or without ouabain. In its sensitivity to ADP, and insensitivity to K+, the phosphopeptide is similar to the E1P of the native enzyme; however, the spontaneous decomposition rate of the phosphopeptide is orders of magnitude lower than that of the native EP. Na+ has no effect on the spontaneous decomposition of the phosphopeptide; but at high Na+ concentrations (K0.5 = 350 mM) the ADP sensitivity of the phosphopeptide is reduced. The phosphopeptide, like the native EP, is acid-stable, alkaline-labile, and sensitive to hydroxylamine and molybdate. The chymotrypsin-treated enzyme catalyzes an ADP-ATP exchange activity that is stimulated by Na+. The Na(+)-independent part of this exchange, unlike that of the native enzyme, is activated by ouabain. Our findings establish that (a) the phosphorylation process and its control by Na+, K+ and ouabain are autoregulated by the NH2-terminal domain of the alpha-subunit; and (b) the often repeated assumption that the primary role of this domain is in the regulation of E1-E2 transitions is not valid.
authors.affiliation: Department of Pharmacology, Medical College of Ohio, Toledo 43699-0008.
authors.forename: W H
authors.initials: WH
authors.lastname: Huang
authors.forename: M
authors.initials: M
authors.lastname: Ganjeizadeh
authors.forename: Y H
authors.initials: YH
authors.lastname: Wang
authors.forename: I N
authors.initials: IN
authors.lastname: Chiu
authors.forename: A
authors.initials: A
authors.lastname: Askari
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D004285:Dogs; D006706:Homeostasis; D007679:Kidney Medulla / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine
chemical_list: D010446:Peptide Fragments; D010710:Phosphates; D010750:Phosphoproteins; D010042:Ouabain; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium