Kinetic Analysis of the Thermostability of Gastric H,K-ATPase

NICOLE T. CERF; VALSECCHI, W.M.; FARAJ, S.E.; MÓNICA R. MONTES; NATALYA FEDOSOVA; ROLANDO C. ROSSI

Congreso; 51st Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq) 46th Congress of Brazilian Biophysical Society (SBBf)/ Lafebs; 2022

INTRODUCTION: The thermostability of an enzyme refers to its ability to remain active and structurally stable after incubation at high temperatures. Yet, due to technical challenges, our understanding of the molecular forces that govern the structure and function of membrane proteins remains underdeveloped in comparison with soluble ones. OBJETIVE: This work presents a kinetic analysis of the thermostability of pig gastric H,K ATPase purified from the apical membrane of the parietal cells. MATERIALS AND METHODS: The enzyme preparation was incubated for different periods and, after cooling, the residual ATPase activity, autophosphorylation capacity and Trp fluorescence was measured. Far UV circular dichroism (CD) spectroscopy was used to examine changes in secondary structure. RESULTS: The inactivation process exhibited first-order kinetics characteristic of a two-state process. We observed parallel time courses for the decrease of ATPase activity, the decrease of autophosphorylation capacity and the loss fluorescence upon heating 49 °C. The correspondence between the kinetics of Trp fluorescence measured at 49 °C and the fluorescence measured after cooling at 25 °C proves the irreversibility of the inactivation process. The native CD spectrum of the H,K ATPase shows two local minima around 209 and 223 nm, compatible with the α/β folding pattern of the protein. Although membranous samples are prone to scatter light, it is possible to appreciate that measurements performed after heating the protein at 49 ºC subtly vary in comparison with the control condition, which was kept at 25 °C throughout the experiment. CONCLUSION: The irreversible loss of tertiary structure reveals the same process as inactivation. However, heating has a marginal effect on secondary structure; such a small change may reflect reversible changes and/or the fact that secondary structure is more resistant than tertiary structure.