title: Effect of Reduction of Redox Modifications of Cys-Residues in the Na,K-ATPase α1-Subunit on Its Activity.
abstract: Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St, 32, Moscow 119991, Russia. dergousova90@list.ru.|Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow 119234, Russia. dergousova90@list.ru.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Dergousova
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St, 32, Moscow 119991, Russia. irina-pva@mail.ru.
authors.forename: Irina Yu
authors.initials: IY
authors.lastname: Petrushanko
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St, 32, Moscow 119991, Russia. klimanova.ea@yandex.ru.|Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow 119234, Russia. klimanova.ea@yandex.ru.
authors.forename: Elizaveta A
authors.initials: EA
authors.lastname: Klimanova
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St, 32, Moscow 119991, Russia. mitkevich@gmail.com.
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Mitkevich
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia. ziganshin@mail.ru.
authors.forename: Rustam H
authors.initials: RH
authors.lastname: Ziganshin
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St, 32, Moscow 119991, Russia. od_lopina@mail.ru.|Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, Moscow 119234, Russia. od_lopina@mail.ru.
authors.forename: Olga D
authors.initials: OD
authors.lastname: Lopina
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St, 32, Moscow 119991, Russia. aamakarov@eimb.ru.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Makarov
mesh_terms: D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004372:Ducks; D005978:Glutathione / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D011489:Protein Denaturation; D021122:Protein Subunits / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D010455:Peptides; D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione; D003545:Cysteine
keywords: Na,K-ATPase; S-glutathionylation; S-nitrosylation; cysteine residues; oxidation; α1-subunit