Dergousova E A, Petrushanko I Yu, Klimanova E A, Mitkevich V A, Ziganshin R H, Lopina O D, Makarov A A
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia.
Faculty of Biology, Moscow State University, Moscow, 119991 Russia.
Mol Biol (Mosk). 2018 Mar-Apr;52(2):289-293. doi: 10.7868/S002689841802012X.
Na,K-ATPase is a transmembrane enzyme that creates a gradient of sodium and potassium, which is necessary for the viability of animal cells. The activity of Na,K-ATPase depends on the redox status of the cell, decreasing with oxidative stress and hypoxia. Previously, we have shown that the key role in the redox sensitivity of Na,K-ATPase is played by the regulatory glutathionylation of cysteine residues of the catalytic alpha subunit, which leads to the inhibition of the enzyme. In this study, the effect of reducing agents (DTT, ME, TCEP) on the level of glutathionylation of the alpha subunit of Na,K-ATPase from rabbit kidneys and the enzyme activity has been evaluated. We have found that the reducing agents partially deglutathionylate the protein, which leads to its activation. It was impossible to completely remove glutathionylation from the native rabbit kidney protein. The treatment of a partially denatured protein on the PVDF membrane with reducing agents (TCEP, NaBH4) also does not lead to the complete deglutathionylation of the protein. The obtained data indicate that Na,K-ATPase isolated from rabbit kidneys has both regulatory and basal glutathionylation, which appears to play an important role in the redox regulation of the function of Na, K-ATPase in mammalian tissues.
钠钾-ATP酶是一种跨膜酶，它能产生钠和钾的梯度，这对动物细胞的存活至关重要。钠钾-ATP酶的活性取决于细胞的氧化还原状态，在氧化应激和缺氧时会降低。此前，我们已经表明，钠钾-ATP酶氧化还原敏感性的关键作用是由催化性α亚基半胱氨酸残基的调节性谷胱甘肽化发挥的，这会导致该酶受到抑制。在本研究中，评估了还原剂（二硫苏糖醇、巯基乙醇、三（2-羧乙基）膦）对兔肾钠钾-ATP酶α亚基谷胱甘肽化水平和酶活性的影响。我们发现，还原剂会使该蛋白部分去谷胱甘肽化，从而导致其激活。无法从天然兔肾蛋白中完全去除谷胱甘肽化。用还原剂（三（2-羧乙基）膦、硼氢化钠）处理聚偏二氟乙烯膜上部分变性的蛋白也不会导致该蛋白完全去谷胱甘肽化。所获得的数据表明，从兔肾中分离出的钠钾-ATP酶具有调节性和基础性谷胱甘肽化，这似乎在哺乳动物组织中钠钾-ATP酶功能的氧化还原调节中发挥着重要作用。
