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The transported cations impose differences in the thermostability of the gastric H,K-ATPase. A kinetic analysis

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  • W. M. Valsecchi, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas
  • ,
  • S. E. Faraj, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas
  • ,
  • N. T. Cerf, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas
  • ,
  • N. U. Fedosova
  • M. R. Montes, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas

This work analyses the thermostability of a membrane protein, the gastric H,K-ATPase, by means of a detailed kinetic characterization of its inactivation process, which showed to exhibit first-order kinetics. We observed parallel time courses for the decrease of ATPase activity, the decrease of the autophosphorylation capacity and the loss of tertiary structure at 49 °C. Higher temperatures were required to induce a significant change in secondary structure. The correspondence between the kinetics of Trp fluorescence measured at 49 °C and the decrease of the residual activity after heating at that temperature, proves the irreversibility of the inactivation process. Inactivation proceeds at different rates in E1 or E2 conformations. The K+-induced E2 state exhibits a lower inactivation rate; the specific effect is exerted with a K0.5 similar to that found at 25 °C, providing a further inkling that K+ occlusion by the H,K-ATPase is not really favoured. Increasing [H+] from pH 8 to pH 7, which possibly shifts the protein to E1, produces a subtle destabilizing effect on the H,K-ATPase. We performed a prediction of potential intramolecular interactions and found that the differential stability between E1 and E2 may be mainly explained by the higher number of hydrophobic interactions in the α- and β-subunits of E2 conformation.

OriginalsprogEngelsk
Artikelnummer184006
TidsskriftBiochimica et Biophysica Acta - Biomembranes
Vol/bind1864
Nummer11
ISSN0005-2736
DOI
StatusUdgivet - 1 nov. 2022

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