Na,K-ATPase reconstituted in ternary liposome: The presence of cholesterol affects protein activity and thermal stability

Abstract

Differential scanning calorimetry (DSC) was applied to investigate the effect of cholesterol on the thermotropic properties of the lipid membrane (DPPC and DPPE). The thermostability and unfolding of solubilized and reconstituted Na,K-ATPase in DPPC:DPPE:cholesterol-liposomes was also studied to gain insight into the role of cholesterol in the Na,K-ATPase modulation of enzyme function and activity. The tertiary system (DPPC:DPPE:cholesterol) (molar ratio DPPC:DPPE equal 1:1) when cholesterol content was increased from 0% up to 40% results in a slight decrease in the temperature of transition and enthalpy, and an increase in width. We observed that, without heating treatment, at 37°C, the activity was higher for 20mol% cholesterol. However, thermal inactivation experiments showed that the enzyme activity loss time depends on the cholesterol membrane content. The unfolding of the enzyme incorporated to liposomes of DPPC:DPPE (1:1mol) with different cholesterol contents, ranging from 0% to 40% mol was also studied by DSC. Some differences between the thermograms indicate that the presence of lipids promotes a conformational change in protein structure and this change is enough to change the way Na,K-ATPase thermally unfolds.

Evaluation of the effect of cafeteria diet on the kidney Na,K‐ATPase activity, and oxidative stress

“…This inhibition promoted by cholesterol is probably related to an increased membrane rigidity, which leads to phospholipid acyl chains "freezing" in the membrane. 42,45 In our study, we noticed that CAF showed a decrease in GSH content in the renal cortex ( Figure 4A) with a decrease in GSH levels and an increase in lipid peroxidation in the renal medulla ( Figure 4E,F, respectively). Among the parameters evaluated in the study by Noeman et al, 8 GSH content was significantly decreased in the renal tissue of the HFD group.…”

“…Another interesting result is shown in Figures C and F, where we demonstrate the relationship between phospholipids and cholesterol. It has been previously demonstrated that the addition of cholesterol to membrane mimetic systems may destabilize the phospholipid bilayer and alter protein conformation . Also, cholesterol concentration appears to have a direct effect on Na,K‐ATPase activity, with variations in its functioning due to its lack or excess in the membrane .…”

“…Moreover, cholesterol has been described to inhibit Na,K‐ATPase activity in supranormal concentrations in studies using membranes of human erythrocytes, guinea pig erythrocytes, rat liver cells, rabbit kidney outer medulla cells and bovine kidney basolateral cells. This inhibition promoted by cholesterol is probably related to an increased membrane rigidity, which leads to phospholipid acyl chains “freezing” in the membrane …”

“…It has been previously demonstrated that the addition of cholesterol to membrane mimetic systems may destabilize the phospholipid bilayer and alter protein conformation. 42 Also, cholesterol concentration appears to have a direct effect on Na,K-ATPase activity, with variations in its functioning due to its lack or excess in the membrane. 43 Besides, 44 demonstrated in erythrocytes that a 20% to 25% depletion in membrane cholesterol led to a reduction in the affinity for internal Na + and an increase in the maximal translocation rate of Na,K-ATPase, while the apparent affinity for external K + was kept intact.…”

Biomedical applications of nanotechnology

“…Proteoliposomes can be used to study how the protein-lipid interactions specifically affect the surface of the membrane model. The scientific community has highlighted this approach as an excellent tool to understand biochemical and biophysical phenomena and to evaluate biotechnological applications (Camolezi et al 2002;Ierardi et al 2002;Daghastanli et al 2004;De Lima Santos et al 2005;Rigos et al 2008;Santos et al 2009;Bolean et al 2010Bolean et al , 2011Bolean et al , 2015Simão et al 2010aSimão et al , 2010bBarbosa et al 2011;Yoneda et al 2013Yoneda et al , 2014Simão et al 2015;Dong et al 2016;Elkhodiry et al 2016). …”