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.

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.

We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the enzyme conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different conformations with different extent of glutathionylation. Incubation of the protein in the E1 conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the enzyme in both E1 and E2 conformations. The pattern of tryptic digestion of the α-subunit in E2 conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 conformation is similar to that of the native enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN conformation than in the E1-and E2-conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects enzyme conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of enzyme glutathionylation and increase in the enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.

Снижение содержания кислорода в тканях, наблюдаемое при целом ряде патологических процессов, неизбежно приводит к их повреждению. Одна из главных причин повреждения и гибели клеток в условиях гипоксии – нарушение работы систем поддержания ионного баланса. Ингибирование активности Na,K-АТРазы, основного ион-транспортирующего белка плазматической мембраны животных клеток, происходящее при снижении концентрации кислорода, считается одним из самых ранних и критичных для жизнеспособности клеток событий. В настоящее время проводится активный поиск модуляторов активности Na,K-АТРазы. Традиционно с этой целью используют сердечные гликозиды, однако вызываемые ими тяжелые побочные эффекты вынуждают искать альтернативные ингибиторы Na,K-АТРазы. Ранее нами было обнаружено, что глутатионилирование каталитической субъединицы Na,K-АТРазы приводит к полному ингибированию фермента. В представленной работе показано, что вещества, увеличивающие уровень глутатионилирования Na,K-АТРазы: этилглутатион (et-GSH), окисленный глутатион (GSSG) и N-ацетилцистеин (NAC), повышают выживаемость клеток в условиях недостатка кислорода, предотвращают падение уровня АТР и нормализуют редокс-статус клеток. Установлен диапазон концентраций, в котором проявляется максимальный защитный эффект этих веществ и отсутствуют выраженные цитотоксические свойства: 0.2–0.5 мМ – у et-GSH, 0.2–1 мМ – у GSSG, 10–15 мМ – у NAC. Полученные результаты показывают перспективность разработки методов защиты тканей от повреждения в условиях кислородного голодания путем изменения степени глутатионилирования Na,K-АТРазы.

Decreasing the amount of oxygen in the tissues under hypoxic and ischemic conditions, observed at a number of pathologic processes, inevitably leads to their damage. One of the main causes of cell damage and death is a violation of the systems maintaining ionic balance. Na,K-ATPaseis a basic ion-transporting protein of animal cell plasma membrane and inhibition of the Na,K-ATPase activity at lower concentrations of oxygen is one of the earliest and most critical events for cell viability. Currently there is an active search for modulators of Na,K-ATPase activity. For this purpose traditionally used cardiac glycosides but the existence of serious adverse effects forced to look for alternative inhibitors of Na,K-ATPase. Previously we have found that the glutathionylation of Na,K-ATPase catalytic subunit leads to a complete-inhibition of the enzyme. In this paper it is shown that the agents which increase the level of Na,K-ATPase glutathionylation: ethyl glutathione (et-GSH), oxidized glutathione (GSSG) and N-acetyl cysteine (NAC), increase cell survival under oxygen deficiency conditions, prevent decline of ATP in the cells and normalize their redox status. Concentration range in which these substances have a maximum protective effect, and does not exhibit cytotoxic properties was defined: for et-GSH 0.2-0.5 mM, for GSSG 0.2-1 mM, for NAC 10 to 15 mM. The results show prospects for development of methods for tissues protection from damage caused by oxygen starvation by varying the degree of Na,K-ATPase glutathionylation.

The decrease in the oxygen content of tissues, which is observed in a number of pathological processes, inevitably leads to damage. One of the main causes of cell damage and death in hypoxia is the failure of the systems that maintain the ionic balance. Na,K-ATPase is the main ion-transporting protein in the plasma membrane of animal cells, and its inhibition at low concentrations of oxygen is one of the earliest and most critical events for cell viability. Scientists are currently conducting an active search for regulators of Na,K-ATPase activity. Cardiac glycosides traditionally used for this purpose induce severe side effects, which necessitates the search for alternative inhibitors of Na,K-ATPase. We have previously found that glutathionylation of the Na,K-ATPase catalytic subunit leads to a complete inhibition of the enzyme. The present study demonstrates that the substances that increase the level of glutathionylation in Na,K-ATPase, namely, ethyl glutathione (et-GSH), oxidized glutathione (GSSG), and N-acetylcysteine (NAC), enhance cell survival under low-oxygen conditions, prevent ATP depletion, and normalize the redox status of the cells. The following concentration range in which these substances have the maximum protective effect and no pronounced cytotoxic properties was determined to be as follows: 0.2–0.5 mM et-GSH, 0.2–1 mM GSSG, and 10–15 mM NAC. These results demonstrate the prospects of developing methods of protecting tissues from damage under low-oxygen conditions that are based on changes in Na,K-ATPase glutathionylation.

A partially purified Na,K-ATPase preparation from rat heart containing α1- and α2-isoforms of the enzyme was shown to include both subunits in S-glutathionylated state. Glutathionylation of the α1-subunit (but not of the α2-subunit) was partially removed when the preparation was isolated in the presence of dithiothreitol. The addition of oxidized glutathione irreversibly inhibited both isoforms. Inhibition of the enzyme containing the α1-subunit was biphasic, and the rate constants of the inhibition were 3745 ± 360 and 246 ± 18 M−1·min−1. ATP, ADP, and AMP protected the Na,K-ATPase against inactivation by oxidized glutathione.

Установлено, что в частично очищенном препарате Na,K-АТРазы из сердца крысы, где присутствуют α1- и α2-изоформы фермента, обе эти субъединицы представлены в S-глутатионилированном состоянии. Глутатионилирование α1- (но не α2-субъединицы) частично устраняется, если препарат выделен в присутствии дитиотреитола. Добавление окисленного глутатиона инактивирует обе изоформы. Инактивация изофермента, содержащего α1-субъединицу, является двухфазной, константы скорости ингибирования составляют 3745 ± 360 и 246 ± 18 М-1 мин-1. ATP, ADP и AMP защищают Na,K-АТРазу сердца от инактивации окисленным глутатионом.