Is antiquitin a mitochondrial Enzyme?

Antiquitin is an aldehyde dehydrogenase involved in the catabolism of lysine. Mutations of antiquitin have been linked with the disease pyridoxine‐dependent seizures. While it is well established that lysine metabolism takes place in the mitochondrial matrix, evidence for the mitochondrial localization of antiquitin has been lacking. In the present study, the subcellular localization of antiquitin was investigated using human embryonic kidney HEK293 cells. Three different approaches were used. First, confocal microscopic analysis was carried out on cells transiently transfected with fusion constructs containing enhanced green fluorescent protein with different lengths of antiquitin based on the different potential start codons of translation. Second, immunofluorescence staining was used to detect the localization of antiquitin directly in the cells. Third, subcellular fractionation was carried out and the individual fraction was analyzed for the presence of antiquitin by Western blot and flow cytometric analyses. All the results showed that antiquitin was present not only in the cytosol but also in the mitochondria. J. Cell. Biochem. 109: 74–81, 2010. © 2009 Wiley‐Liss, Inc.     

Is HCO3? Transport in Anabaena a Na+ Symport?

Na⁺ strongly promoted HCO₃⁻ transport in Anabaena variabilis. The effect was highly specific to this cation. Kinetic analysis indicated a progressive decrease in the K_m (HCO₃⁻) of the transport system with increasing Na⁺ concentration. V_max was also affected. We raise the possibility that the transport is a Na⁺-HCO₃⁻ symport; alternatively, that a Na⁺-H⁺ antiport (or Na⁺-OH⁺ symport) system mediates the efflux of the OH⁻ ions derived from the entering HCO₃⁻ ions, and that this antiport can rate-limit HCO₃⁻ influx.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.     

Polarographic studies of membrane particles containing Na−K ATPase

Summary The properties of a suspension of membrane particles containing Na–K ATPase have been investigated with the aid of d–c and a–c polarography. In particular, we have studied the interaction of three cations, two very effective enzyme inhibitors and one activator, with the enzyme preparation. Ag⁺ and Cu⁺⁺, which inhibit the enzyme at very low concentrations, bind very strongly. No binding could be found with the activating ion, Tl⁺, however. Adsorption of a substance with an isoelectric point between pH 4 and pH 5.5 occurred at the electrode surface between –0.1 and –1.2 V at pH 7, and was associated with the random currents that appear during the measurements. The random currents arise when the membrane particles collide with the electrode and cause changes in the structure of the electrical double layer. (Added substances that adsorb more strongly at the mercury/water interface eliminate the random currents.) The adsorbed film impedes the flow of the free Ag⁺ and Cu⁺⁺ ions, and to a smaller extent, the flow of Tl⁺ ions. The differences between the binding of inhibiting and activating ions are correlated with their effects on the ATPase enzyme activity.     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.     

HCO 3 − -coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

Summary Recent studies in hepatocytes indicate that Na⁺-coupled HCO ₃ ^– transport contributes importantly, to regulation of intracellular pH and membrane HCO ₃ ^– transport. However, the direction of net coupled Na⁺ and HCO ₃ ^– movement and the effect of HCO ₃ ^– on Na⁺ turnover and Na⁺/K⁺ pump activity are not known. In these studies, the effect of HCO ₃ ^– on Na⁺ influx and turnover were measured in primary rat hepatocyte cultures with²²Na⁺, and [Na⁺] _i was measured in single hepatocytes using the Na⁺-sensitive fluorochrome SBFI. Na⁺/K⁺ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na⁺-dependent or ouabain-suppressible⁸⁶Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K⁺ on membrane potential difference (PD) and [Na⁺] _i . In hepatocyte monolayers, HCO ₃ ^– increased²²Na⁺ entry and turnover rates by 50–65%, without measurably altering²²Na⁺ pool size or cell volume, and HCO ₃ ^– also increased Na⁺/K⁺ pump activity by 70%. In single cells, exposure to HCO ₃ ^– produced an abrupt and sustained rise in [Na⁺] _i , from 8 to 12mm. Na⁺/K⁺ pump activity assessed in single cells by PD excursions during transient K⁺ removal increased 2.5-fold in the presence of HCO ₃ ^– , and the rise in [Na⁺] _i produced by inhibition of the Na⁺/K⁺ pump was similarly increased 2.5-fold in the presence of HCO ₃ ^– . In intact perfused rat liver, HCO ₃ ^– increased both Na⁺/K⁺ pump activity and O₂ consumption. These findings indicate that, in hepatocytes, net coupled Na⁺ and HCO ₃ ^– movement is inward and represents a major determinant of Na⁺ influx and Na⁺/K⁺ pump activity. About half of hepatic Na⁺/K⁺ pump activity appears dedicated to recycling Na⁺ entering in conjunction with HCO ₃ ^– to maintain [Na⁺] _i within the physiologic range.     

S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.     

Structural determinants for the ouabain-stimulated increase in Na–K ATPase activity

Recent studies suggest that at low concentrations, ouabain increases Na–K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na–K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1 μg kg body wt^− 1 day^− 1) for 9 days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na–K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of ⁸⁶Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na–K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na–K ATPase α1 is required for translocation of both Na–K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na–K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na–K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney

A study has been made to determine whether renal plasma membranes contain an HCO₃^? stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney.The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase.The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome $\text{c}$ oxidase activity.These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Do H+ ions obscure electrogenic Na+ and K+ binding in the E1 state of the Na,K-ATPase?

In contrast to other P-type ATPases, the Na,K-ATPase binding and release of ions on the cytoplasmic side, to the state called E1, is not electrogenic with the exception of the third Na+. Since the high-resolution structure of the closely related SR Ca-ATPase in state E1 revealed the ion-binding sites deep inside the transmembrane part of the protein, the missing electrogenicity in state E1 can be explained by an obscuring counter-movement of H+ ions. Evidence for such a mechanism is presented by analysis of pH effects on Na+ and K+ binding and by electrogenic H+ movements in the E1 conformation of the Na,K-ATPase.     

Diabetes induced by streptozotocin causes reduced Na−K ATPase in the brain

Na–K ATPase activity in the brain decreased significantly after diabetes was induced with streptozotocin in rats. Largest decreases were observed in the hippocampus (–30%) and the cerebral cortex (–26%). Smaller decreases were observed in the thalamus (–13%), hypothalamus (–11%) and brain stem (–10%). Na–K ATPase activity in the striatum and the cerebellum were not significantly decreased. The varied decreases suggest that the regional variation of the enzyme is enhanced in the diabetic state. The enzymes of glucose metabolic pathway, namely hexokinase, lactate dehydrogenase and citrate synthase in the brain regions largely remained unchanged although increases in lactate dehydrogenase were observed in some regions. Acetylcholinesterase activity, a marker for the cholinergic system, remains unaltered in the brain during diabetes. The results are discussed with respect to the possible metabolic factors which alter the Na–K ATPase in the brain and its comparison with the peripheral nerve.     

Cardiotonic steroids as potential Na+/K+-ATPase inhibitors – a computational study

Abstract

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na⁺/K⁺-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na⁺/K⁺-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470?kcal/mol to 7.234?kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470?kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na⁺/K⁺-ATPase-ouabain and Na⁺/K⁺-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100?ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.     

Is ryanodine receptor a calcium or magnesium channel? Roles of K+ and Mg2+ during Ca2+ release

The ryanodine receptor (RyR) is a poorly selective channel that mediates Ca(2+) release from intracellular Ca(2+) stores. How RyR's selectivity between the physiological cations K(+), Mg(2+), and Ca(2+) affects single-channel Ca(2+) current amplitude is examined using a recent model of RyR permeation. It is found that K(+) provides the vast majority of the countercurrent (through RyR itself) that is needed to prevent the sarcoplasmic reticulum (SR) membrane potential from changing and stopping Ca(2+) release. Moreover, intra-pore competition between Ca(2+) and Mg(2+) defines single RyR Ca(2+) current amplitude. Since both [Mg(2+)] and [Ca(2+)](SR) can change during pathophysiological conditions, the RyR unitary Ca(2+) current amplitude during Ca(2+) release may change significantly due to this Ca(2+)/Mg(2+) competition. Compared to the classic action of Mg(2+) on RyR open probability, these Ca(2+) current amplitude changes have as large or larger effects on overall RyR Ca(2+) mobilization. A new aspect of RyR divalent versus monovalent selectivity is also identified where this kind of selectivity decreases as divalent concentration increases.     

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na⁺-K⁺-2Cl^– cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl^– depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl^– depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC. proline-alanine-rich STE20-related kinase     

Enzyme with a memory for its substrate?

<正>In the interaction between an enzyme and its substrate,the enzyme is induced by the substrate followed by a change in enzymic conformation to fit the substrate.So far,many laboratories have been studying the neural memory.Recently,