Abstract
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.
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Abbreviations
- ME:
-
β-mercaptoethanol
- DTT:
-
dithiothreitol
- TCEP:
-
Tris(2-carboxyethyl)phosphine hydrochloride
- EDTA:
-
ethylenediaminetetraacetic acid
- PBST:
-
phosphate-buffered saline supplemented with Tween-20
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Original Russian Text © E.A. Dergousova, I.Yu. Petrushanko, E.A. Klimanova, V.A. Mitkevich, R.H. Ziganshin, O.D. Lopina, A.A. Makarov, 2018, published in Molekulyarnaya Biologiya, 2018, Vol. 52, No. 2, pp. 289–293.
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Dergousova, E.A., Petrushanko, I.Y., Klimanova, E.A. et al. Enhancement of Na,K-ATPase Activity as a Result of Removal of Redox Modifications from Cysteine Residues of the α1 Subunit: the Effect of Reducing Agents. Mol Biol 52, 247–250 (2018). https://doi.org/10.1134/S0026893318020024
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DOI: https://doi.org/10.1134/S0026893318020024