title: Opposing effects of Na+ and K+ on the thermal stability of Na+,K(+)-ATPase.
abstract: Folding and structural stability are key factors for the proper biological function of proteins. Na(+),K(+)-ATPase is an integral membrane protein involved in the active transport of Na(+) and K(+) across the plasma membrane. In this work we characterized the effects of K(+) and Na(+) on the thermal inactivation of Na(+),K(+)-ATPase, evaluating both catalytic and transport capacities of the pump. Both activities of the enzyme decrease with the preincubation time as first-order kinetics. The thermal inactivation of Na(+),K(+)-ATPase is simultaneous with a conformational change detected by tryptophan and 1-aniline-8-naphtalenesulfonate (ANS) fluorescence. The kinetic coefficient of thermal inactivation was affected by the presence of Na(+) and K(+) (or Rb(+)) and the temperature of the preincuabtion media. Our results show that K(+) or Rb(+) stabilize the enzyme, while Na(+) decreases the stability of Na(+),K(+)-ATPase. Both effects are exerted by the specific binding of these cations to the pump. Also, we provided strong evidence that the Rb(+) (or K(+)) stabilization effect is due to the occlusion of these cations into the enzyme. Here, we proposed a minimal kinetic model that explains the behavior observed in the experimental results and allows a better understanding of the results presented by other researchers. The thermal inactivation process was also analyzed according to Kramer's theory.
authors.affiliation: Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Sergio B
authors.initials: SB
authors.lastname: Kaufman
authors.forename: F Luis
authors.initials: FL
authors.lastname: González-Flecha
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: González-Lebrero
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002412:Cations / Q000737:chemistry; D007700:Kinetics; D011188:Potassium / Q000737:chemistry*; D055550:Protein Stability; D012413:Rubidium / Q000737:chemistry; D012964:Sodium / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000817:Anilino Naphthalenesulfonates; D002412:Cations; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium