Yoneda Juliana Sakamoto, Rigos Carolina Fortes, de Lourenço Thaís Fernanda Aranda, Sebinelli Heitor Gobbi, Ciancaglini Pietro
Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil.
Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - FFCLRP-USP, Depto. Química, 14040-901 Ribeirão Preto, SP, Brazil.
Arch Biochem Biophys. 2014 Dec 15;564:136-41. doi: 10.1016/j.abb.2014.09.015. Epub 2014 Oct 5.
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.
采用差示扫描量热法（DSC）研究胆固醇对脂质膜（二棕榈酰磷脂酰胆碱和二棕榈酰磷脂酰乙醇胺）热致性质的影响。还研究了溶解并重构于二棕榈酰磷脂酰胆碱：二棕榈酰磷脂酰乙醇胺：胆固醇脂质体中的钠钾-ATP酶的热稳定性和去折叠情况，以深入了解胆固醇在钠钾-ATP酶调节酶功能和活性中的作用。当胆固醇含量从0%增加到40%时，三元体系（二棕榈酰磷脂酰胆碱：二棕榈酰磷脂酰乙醇胺：胆固醇）（二棕榈酰磷脂酰胆碱与二棕榈酰磷脂酰乙醇胺的摩尔比为1:1）导致转变温度和焓略有降低，宽度增加。我们观察到，未经加热处理时，在37°C下，胆固醇含量为20mol%时活性较高。然而，热失活实验表明，酶活性丧失时间取决于胆固醇膜含量。还通过DSC研究了掺入不同胆固醇含量（0%至40%摩尔）的二棕榈酰磷脂酰胆碱：二棕榈酰磷脂酰乙醇胺（1:1摩尔）脂质体中的酶的去折叠情况。热谱图之间的一些差异表明，脂质的存在促进了蛋白质结构的构象变化，这种变化足以改变钠钾-ATP酶的热去折叠方式。
