RESUMO
Melittin, a peptide from bee venom, was found to be able to interact with many proteins, including calmodulin target proteins and ion-transporting P-type ATPases. It is assumed that melittin mimics a protein module involved in protein-protein interactions within cells. Previously, a Na^(+)/K^(+)-ATPase containing the α1 isoform of the catalytic subunit was found to co-precipitate with a protein with a molecular weight of about 70 κDa that interacts with antibodies against melittin by cross immunoprecipitation. In the presence of a specific Na^(+)/K^(+)-ATPase inhibitor (ouabain), the amount of protein with a molecular weight of 70 κDa interacting with Na^(+)/K^(+)-ATPase increases. In order to identify melittin-like protein from murine kidney homogenate, a fraction of melittin-like proteins with a molecular weight of approximately 70 κDa was obtained using affinity chromatography with immobilized antibodies specific to melittin. By mass spectrometry analysis, the obtained protein fraction was found to contain three molecular chaperones of Hsp70 superfamily: mitochondrial mtHsp70 (mortalin), Hsp73, Grp78 (BiP) of endoplasmic reticulum. These data suggest that chaperones from the HSP-70 superfamily contain a melittin-like module.
Assuntos
Meliteno , ATPase Trocadora de Sódio-Potássio , Camundongos , Animais , Meliteno/química , Meliteno/metabolismo , Meliteno/farmacologia , ATPase Trocadora de Sódio-Potássio/química , Peso Molecular , Ouabaína/farmacologia , Peptídeos/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+ i/K+ i-dependent mechanism(s).
RESUMO
Long-term study on the identification of Na,K-ATPase endogenous inhibitors in mammalian tissues has resulted in the discovery of ouabain, marinobufagenin (MBG), and other cardiotonic steroids (CTS) in the blood plasma. Production of ouabain and MBG is increased in essential hypertension and other diseases associated with hypervolemia. Here, we compared the effects of ouabain and MBG on the Na,K-ATPase activity (measured as the transport of Na+, K+, and Rb+ ions) and proliferation and death of human renal epithelial cells (HRECs) and human umbilical vein endothelial cells (HUVEC) expressing α1-Na,K-ATPase. Ouabain concentration that provided the half-maximal inhibition of the Rb+ influx (IC50) into HRECs and HUVECs was 0.07 µM. In both types of cells, the IC50 values for MBG were 10 times higher than for ouabain. Incubation of HREC and HUVEC with 0.001-0.01 µM ouabain for 30 h resulted in 40% increase in the [3H]thymidine incorporation into DNA; further elevation of ouabain concentration to 0.1 µM completely suppressed DNA synthesis. MBG at the concentration of 0.1 µM activated DNA synthesis by 25% in HRECs, but not in HUVECs; 1 µM MBG completely inhibited DNA synthesis in HRECs and by 50% in HUVECs. In contrast to HRECs, incubation of HUVECs in the serum-free medium induced apoptosis, which was almost completely suppressed by ouabain and MBG at the concentrations of 0.1 and 3 µM, respectively. Based on these data, we can conclude that (i) the effect of MBG at the concentrations detected in the blood plasma (<0.01 µM) on HRECs and HUVECs was not due to the changes in the [Na+]i/[K+]i ratio; (ii) the effect of physiological concentrations of ouabain on these cells might be mediated by the activation of Na,K-ATPase, leading to cell proliferation.
Assuntos
Bufanolídeos/farmacologia , Proliferação de Células , Células Endoteliais/fisiologia , Células Epiteliais/fisiologia , Coração/fisiologia , Ouabaína/farmacologia , Cardiotônicos/farmacologia , Morte Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Coração/efeitos dos fármacos , Humanos , Transporte de Íons , Vasoconstritores/farmacologiaRESUMO
Maintenance of non-equilibrium Na+ and K+ distribution between cytoplasm and extracellular medium suggests existence of sensors responding with conformational transitions to the changes of these monovalent cations' intracellular concentration. Molecular nature of monovalent cation sensors has been established in Na,K-ATPase, G-protein-coupled receptors, and heat shock proteins structural studies. Recently, it was found that changes in Na+ and K+ intracellular concentration are the key factors in the transcription and translation control, respectively. In this review, we summarize results of these studies and discuss physiological and pathophysiological significance of Na+i,K+i-dependent gene expression regulation mechanism.
Assuntos
Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Animais , Cátions Monovalentes/química , Citoplasma/metabolismo , Proteínas de Choque Térmico/metabolismo , Potássio/química , Biossíntese de Proteínas , Sódio/química , Transcrição GênicaRESUMO
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 modeling 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, glutathionylation 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.
Assuntos
Glutationa/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteólise , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Tripsina/metabolismo , Animais , Relação Dose-Resposta a Droga , Patos , Modelos Moleculares , Ouabaína/farmacologia , Cloreto de Potássio/farmacologia , Conformação Proteica , Proteólise/efeitos dos fármacos , Cloreto de Sódio/farmacologiaRESUMO
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.
Assuntos
ATPase Trocadora de Sódio-Potássio/química , Animais , Cisteína/química , Cisteína/metabolismo , Ativação Enzimática , Oxirredução , Coelhos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Participation of Na+/K+-ATPase in the natriuretic effect of prolactin in a cholestasis of pregnancy model was investigated. The Na+/K+-ATPase activity in rat kidney medulla, where active sodium reabsorption occurs, decreased in the model of cholestasis of pregnancy and other hyperprolactinemia types compared with intact animals. This effect was not connected with the protein level of α1- and ß-subunits of Na+/K+-ATPase measured by Western blotting in the kidney medulla. Decrease in Na+/K+-ATPase activity in the kidney cortex was not significant, as well as decrease in the quantity of mRNA and proteins of the α1- and ß-subunits of Na+/K+-ATPase. There were no correlations between the Na+/K+-ATPase activity and sodium clearance, although sodium clearance increased significantly in the model of cholestasis of pregnancy and other hyperprolactinemia groups under conditions of stable glomerular filtration rate measured by creatinine clearance. We conclude that the Na+/K+-ATPase is not the only mediator of the natriuretic effect of prolactin in the model of cholestasis of pregnancy.
Assuntos
Colestase/urina , Medula Renal/metabolismo , Complicações na Gravidez/urina , Prolactina/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/urina , Animais , Colestase/induzido quimicamente , Modelos Animais de Doenças , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Gravidez , Complicações na Gravidez/induzido quimicamente , RatosRESUMO
Prolonged exposure of different epithelial cells (canine renal epithelial cells (MDCK), vascular endothelial cells from porcine aorta (PAEC), human umbilical vein endothelial cells (HUVEC), cervical adenocarcinoma (HeLa), as well as epithelial cells from colon carcinoma (Caco-2)) with ouabain or with other cardiotonic steroids was shown earlier to result in the death of these cells. Intermediates in the cell death signal cascade remain unknown. In the present study, we used proteomics methods for identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain. After exposure of Caco-2 human colorectal adenocarcinoma cells with 3 µM of ouabain for 3 h, the protein interacting in complex with Na+,K+-ATPase was coimmunoprecipitated using antibodies against the enzyme α1-subunit. Proteins of coimmunoprecipitates were separated by 2D electrophoresis in polyacrylamide gel. A number of proteins in the coimmunoprecipitates with molecular masses of 71-74, 46, 40-43, 38, and 33-35 kDa was revealed whose binding to Na+,K+-ATPase was activated by ouabain. Analyses conducted by mass spectroscopy allowed us to identify some of them, including seven signal proteins from superfamilies of glucocorticoid receptors, serine/threonine protein kinases, and protein phosphatases 2C, Src-, and Rho-GTPases. The possible participation of these proteins in activation of cell signaling terminated by cell death is discussed.
Assuntos
Ouabaína/farmacologia , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células CACO-2 , Morte Celular/efeitos dos fármacos , Cães , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Madin Darby de Rim Canino , SuínosRESUMO
Side-by-side with inhibition of the Na+,K+-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na+ and K+ ratio ([Na+]i/[K+]i). Thus, we compared the dose- and time-dependences of the effect of ouabain on intracellular [Na+]i/[K+]i ratio, Na+,K+-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na+]i/[K+]i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na+,K+-ATPase activity by 25-30%, as measured by the rate of (86)Rb(+) influx. Higher ouabain concentrations inhibited Na+,K+-ATPase, increased [Na+]i/[K+]i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na+]i/[K+]i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na+]i/[K+]i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na+ and K+ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na+,K+-ATPase and decrease in the [Na+]i/[K+]i ratio.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Ouabaína/farmacologia , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Transporte de Íons/efeitos dos fármacosRESUMO
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.
Assuntos
Meliteno/metabolismo , Peptídeos/análise , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Imunoprecipitação , Meliteno/química , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Coelhos , ATPase Trocadora de Sódio-Potássio/química , Tripsina/metabolismoRESUMO
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.
Assuntos
Glutationa/metabolismo , Isquemia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Acetilcisteína/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Domínio Catalítico/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glutationa/química , Dissulfeto de Glutationa/farmacologia , Humanos , Isquemia/tratamento farmacológico , Isquemia/patologia , Camundongos , Consumo de Oxigênio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
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.
Assuntos
Dissulfeto de Glutationa/metabolismo , Dissulfeto de Glutationa/farmacologia , Miocárdio/enzimologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , Animais , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ratos , Ratos WistarRESUMO
Isotopic effects of deuterium in water are studied in a broad range of concentrations on a number of biological objects of different organization levels. The results obtained show that biological objects are sensitive to variations of isotope composition in water. A decrease or increase in deuterium concentrations in water may cause activation or inhibition of biological functions. The values of biological isotopic effects of low deuterium concentration may even be higher than those of high deuterium concentration. No regularity in response for all the objects studied failed to find out in a range of deuterium concentration in water from 4 ppm to 1%.
Assuntos
Óxido de Deutério/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Germinação/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Gastrópodes , Humanos , Lepidium/efeitos dos fármacos , Lepidium/fisiologia , Masculino , Rana temporaria , Triticum/efeitos dos fármacos , Triticum/fisiologiaRESUMO
Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (~100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (~67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.
Assuntos
Medula Renal/enzimologia , Neprilisina , ATPase Trocadora de Sódio-Potássio , Animais , Hidrólise , Neprilisina/química , Neprilisina/isolamento & purificação , Neprilisina/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Coelhos , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Ouabain and other cardiotonic steroids (CTS) kill renal epithelial cells from distal tubules (C7-MDCK) via interaction with Na,K-ATPase but independently of inhibition of Na,K-ATPase-mediated ion fluxes. Recently, we demonstrated that modest intracellular acidification and inhibition of p38 MAPK suppress death of C7-MDCK cells triggered by ouabain. In the present study we investigate the mechanism of p38 MAPK activation in renal epithelial cell from distal tubules evoked by cardiotonic steroids. Using Na+/K+ ionophores (monensin, nigericin) and media with different content of monovalent cations, we revealed that p38 MAPK phosphorylation in ouabain-treated renal epithelial cells is not caused by Na,K-ATPase inhibition and inversion of the [Na+](i)/[K+](i) ratio. We also demonstrated that attenuation of pH from 7.45 to 6.75 did not alter the level of p38 MAPK phosphorylation observed in ouabain-treated cells. Inhibitors of PKA, PKC, and PKG as well as protein phosphatases were unable to abolish p38 MAPK activation triggered by ouabain. Using phosphotyrosine antibodies we did not detect any effect of ouabain on activation of tyrosine kinases. Thus, our results show that activation of p38 MAPK and cytotoxic action of CTS are independent of intracellular Na+, K+, and H+ concentrations. The molecular origin of intermediates of death signaling induced by CTS via conformation changes of Na,K-ATPase with following activation of p38 MAPK should be examined further.
Assuntos
Glicosídeos Cardíacos/farmacologia , Células Epiteliais/enzimologia , Túbulos Renais Distais/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Morte Celular , Linhagem Celular , Cães , Células Epiteliais/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Túbulos Renais Distais/efeitos dos fármacos , Ouabaína/farmacologia , Fosforilação , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8-2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase α-subunit phosphorylation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane microviscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in membranes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.
Assuntos
Rim/metabolismo , Microssomos/enzimologia , Sciuridae/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Hibernação , Isoenzimas/metabolismo , Multimerização Proteica , Estações do AnoRESUMO
Na+/K+-ATPase generates an electrochemical gradient of Na+ and K+, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two ground conformational states, E1 and E2. To characterize the domain organization of the protein in these conformations, the thermal denaturation of Na+/K+-ATPase from duck salt glands and rabbit kidneys has been studied in the presence of Na+ and K+, which induce the transition of the enzyme to the conformation E1 or E2. The melting curves for the apoforms of Na+/K+-ATPases have different shapes: the curve for the enzyme from the rabbit shows one transition at 56.1 degrees C, whereas the denaturation of Na+/K+-ATPase from the duck is characterized by two transitions, at 49.8 and 56.9 degrees C. Sodium and potassium ions abolish the difference in the domain organization of Na+/K+-ATPases. The melting curves for Na+/K+-ATPases in conformation E2 in both cases exhibit a single peak of thermal absorption at about 63 degrees C. The melting curves for the enzymes in conformation E1 show three peaks of thermal absorption, indicating the denaturation of three domains. The difference in the domain organization of Na+/K+-ATPase in conformations E1 and E2 may be of importance in different sensitivity of these conformations of the enzyme to temperature, proteolytic enzymes, and oxidative stress.
Assuntos
ATPase Trocadora de Sódio-Potássio/química , Animais , Apoproteínas/química , Cátions , Patos , Temperatura Alta , Isoenzimas/química , Rim/enzimologia , Potássio/química , Conformação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Coelhos , Glândula de Sal/enzimologia , Sódio/química , Especificidade da EspécieRESUMO
Low molecular mass proteins of the FXYD family that affect the sensitivity of Na,K-ATPase to Na+ and K+ are known to be present in Na,K-ATPases in various tissues. In particular, in Na,K-ATPase from kidney a gamma-subunit (with electrophoretic mobility corresponding to molecular mass of about 10 kD) is present, and Na,K-ATPase preparations from heart contain phospholemman (electrophoretic mobility of this protein corresponds to molecular mass of 13-14 kD), which provides for the interaction of heart Na,K-ATPase with cytoskeletal microtubules. Disruption of microtubules by colchicine removes phospholemman from heart Na,K-ATPase preparations. The goal of the present study was to reveal a low molecular mass protein (probably a member of FXYD family) in preparation of Na,K-ATPase from duck salt glands. Immunoprecipitation of solubilized duck salt gland Na,K-ATPase using antibodies against alpha1-subunit results in the coprecipitation of a 13 kD protein with the Na,K-ATPase complex. Treatment of homogenate from duck salt glands with colchicine removes this protein from the purified preparation of Na,K-ATPase. Simultaneously, we observed a decrease in the sensitivity of Na,K-ATPase to Na+ at pH 6.5. However, colchicine treatment of homogenate from rabbit kidney does not affect either the sensitivity of Na,K-ATPase obtained from this homogenate to Na+ or the content of 10 kD protein (presumably gamma-subunit). The data suggest that phospholemman (or a similar member of the FXYD family) tightly interacts with Na,K-ATPase from duck salt glands and binds it to microtubules, simultaneously participating in the regulation of the sensitivity of Na,K-ATPase to Na+.
Assuntos
Colchicina/farmacologia , Patos/metabolismo , Glândula de Sal/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia , Animais , Colchicina/metabolismo , Imunoprecipitação , Potássio/metabolismo , Subunidades Proteicas/metabolismo , Coelhos , Glândula de Sal/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.
Assuntos
Anticorpos/química , Inibidores Enzimáticos/química , Meliteno/química , Ouabaína/química , ATPase Trocadora de Sódio-Potássio/química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Domínio Catalítico/imunologia , Inibidores Enzimáticos/farmacologia , Meliteno/genética , Meliteno/imunologia , Camundongos , Peso Molecular , Ouabaína/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Coelhos , Ratos , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/imunologia , SuínosRESUMO
Review of hormonal function of endogenous cardiac steroids. Special attention is paid to recently discovered mechanism of signal transduction from Na,K-ATPase that is due to not a change of ionic gradients but to ouabain-induced alteration of enzyme conformation, that, in turn, results in interaction of the enzyme with intracellular proteins. The data concerning discovery and identification of endogenous cardiac steroids and different isoforms of Na,K-ATPase that have various sensitivity to cardiac steroid, are also considered.