title: 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.
authors.affiliation: Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Juliana Sakamoto
authors.initials: JS
authors.lastname: Yoneda
authors.affiliation: Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Carolina Fortes
authors.initials: CF
authors.lastname: Rigos
authors.affiliation: Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Thaís Fernanda Aranda
authors.initials: TF
authors.lastname: de Lourenço
authors.affiliation: Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil.
authors.forename: Heitor Gobbi
authors.initials: HG
authors.lastname: Sebinelli
authors.affiliation: Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil. Electronic address: pietro@ffclrp.usp.br.
authors.forename: Pietro
authors.initials: P
authors.lastname: Ciancaglini
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D008081:Liposomes / Q000737:chemistry*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D058767:Protein Unfolding; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008081:Liposomes; D010714:Phosphatidylethanolamines; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Detergent solubilized; Differential scanning calorimetry; Na,K-ATPase; Proteoliposome; Thermal inactivation; Unfolding