Protective Effect Of Na Research Articles

A Protective Strategy to Counteract the Oxidative Stress Induced by Simulated Microgravity on H9C2 Cardiomyocytes.

Microgravity affects human cardiovascular function inducing heart rhythm disturbances and even cardiac atrophy. The mechanisms triggered by microgravity and the search for protection strategies are difficult to be investigated in vivo. This study is aimed at investigating the effects induced by simulated microgravity on a cardiomyocyte-like phenotype. The Random Positioning Machine (RPM), set in a CO2 incubator, was used to simulate microgravity, and H9C2 cell line was used as the cardiomyocyte-like model. H9C2 cells were exposed to simulated microgravity up to 96 h, showing a slower cell proliferation rate and lower metabolic activity in comparison to cell grown at earth gravity. In exposed cells, these effects were accompanied by increased levels of intracellular reactive oxygen species (ROS), cytosolic Ca2+, and mitochondrial superoxide anion. Protein carbonyls, markers of protein oxidation, were significantly increased after the first 48 h of exposition in the RPM. In these conditions, the presence of an antioxidant, the N-acetylcysteine (NAC), counteracted the effects induced by the simulated microgravity. In conclusion, these data suggest that simulated microgravity triggers a concomitant increase of intracellular ROS and Ca2+ levels and affects cell metabolic activity which in turn could be responsible for the slower proliferative rate. Nevertheless, the very low number of detectable dead cells and, more interestingly, the protective effect of NA, demonstrate that simulated microgravity does not have “an irreversible toxic effect” but, affecting the oxidative balance, results in a transient slowdown of proliferation.

The protective effect of Na+/Ca2+ exchange blocker kb-r7943 on myocardial ischemia-reperfusion injury in hypercholesterolemic rat.

KB-R7943 reduces lethal reperfusion injury under normal conditions, but its effectiveness under certain pathological states is in dispute. In the present study, we sought to determine the effect of KB-R7943 in hyperlipidemic animals and assess if the K ATP (+) are involved in the protective mechanisms. In group 1 (G1), isolated rat hearts underwent 25 min global ischemia (GI) and 120 min reperfusion (R). In group 2 (G2), G1 was repeated but the animals were subjected to a 1.5 % cholesterol-enriched diet during 6 weeks (hypercholesterolemic animals). In group 3 (G3), G2 was repeated but 1 μM KB-R7943 was added to the perfusate for 10 min from the start of reperfusion. In group 4 (G4), G3 was repeated, and glibenclamide (K ATP (+) , blocker, 0.3 μM) was administered. The infarct size was measured by triphenyltetrazolium. The infarct size was 35 ± 5.0 % in G1 and 46 ± 8.7 % in G2 (P < 0.05); KB-R7943 reduced the infarct size (28.6 ± 3.3 % in G3 vs. G2, P < 0.05). In addition, KB-R7943 attenuated apoptotic cell (G3 vs. G2, P < 0.05), but glibenclamide abolished the effect reached by KB-R7943. Thus, diet-induced hypercholesterolemia enhances myocardial injury; KB-R7943 reduces infarct size and apoptosis in hyperlipidemic animals through the activation of K(+)ATP channels.

Metabolomic tissue signature in human non‐alcoholic fatty liver disease identifies protective candidate metabolites

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries, yet its pathophysiology is incompletely understood. Small-molecule metabolite screens may offer new insights into disease mechanisms and reveal new treatment targets. Discovery (N = 33) and replication (N = 66) of liver biopsies spanning the range from normal liver histology to non-alcoholic steatohepatitis (NASH) were ascertained ensuring rapid freezing under 30 s in patients. 252 metabolites were assessed using GC/MS. Replicated metabolites were evaluated in a murine high-fat diet model of NAFLD. In a two-stage metabolic screening, hydroquinone (HQ, p(combined) = 3.0 × 10(-4)) and nicotinic acid (NA, p(combined) = 3.9 × 10(-9)) were inversely correlated with histological NAFLD severity. A murine high-fat diet model of NAFLD demonstrated a protective effect of these two substances against NAFLD: Supplementation with 1% HQ reduced only liver steatosis, whereas 0.6% NA reduced both liver fat content and serum transaminase levels and induced a complex regulatory network of genes linked to NALFD pathogenesis in a global expression pathway analysis. Human nutritional intake of NA equivalent was also consistent with a protective effect of NA against NASH progression. This first small-molecular screen of human liver tissue identified two replicated protective metabolites. Either the use of NA or targeting its regulatory pathways might be explored to treat or prevent human NAFLD.

Protective effect of ions against cell death induced by acid stress inSaccharomyces

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na(+) in yeast viability in acidic conditions was tested using S. cerevisiae Na(+)-ATPases (ena1-4), Na(+)/H(+) antiporter (nha1Delta) and Na(+)/H(+) antiporter prevacuolar (nhx1Delta) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells.

The combined treatment of Na+/H+ exchange inhibitor and β‐blocker additively protects mitochondria following cardiac ischemic reperfusion injury

The protective effect of Na+/H+ exchange inhibitor (NHE) has been well documented on experimental studies. However clinical trials failed to show beneficial effects by either improving clinical outcome or limiting infarct size. In this study we investigated whether the combination of NHE Cariporide (Car) and β-blocker Metoprolol (Met) additively protect mitochondria (mito) following cardiac ischemic reperfusion (I/R). Isolated hearts from Sprague-Dawley rats were subjected to 20 min of global ischemia followed by 60 min reperfusion and divided into Control (Cont) and 10 min drug pretreatments of Car (10μM), Met (10μM) and Car+Met respectively. At the end of reperfusion each group of hearts were randomized for infarct size determination, left ventricle mito Ca2+ and respiratory control ratio (RCR) analyses (Table *p<0.05 vs. Cont, †p<0.05 vs. Car and Met). Although Car+Met did not improve mito protein yield (data not shown), it decreased mito Ca2+ content, increased RCR and limited infarct size. These results suggested that this combined treatment additively increases the protection of mitochondria and limits the infarct size following I/R. This strategy may make the treatment with Na+/H+ exchange inhibitor becoming practical for clinical use.

Na/H exchange inhibition in hypertrophied myocardium subjected to cardioplegic arrest: an effective cardioprotective approach

This study was designed to assess whether the protective effects of Na+/H+ exchange (NHE) inhibition, which have been largely demonstrated in normal hearts, are also manifest in a more surgically relevant model of hypertrophied myocardium subjected to cardioplegic arrest. Left ventricular hypertrophy was created in 3-week-old rats by coarctation of the ascending thoracic aorta with a hemoclip. Eight weeks later, hearts were excised, isovolumetrically perfused and subjected to 1 h of potassium cardioplegic arrest followed by 2 h of reperfusion. Hearts were allocated to one of the following four groups: sham-operated and aortic banding hearts without any treatment or treated with the NHE inhibitor cariporide (1 micromol/L) given as an additive to cardioplegia and over the first 15 min of reperfusion. The major effect of cariporide was to reduce ischemic peak contacture and to improve post-ischemic diastolic function in both sham-operated and hypertrophied hearts. Total creatine kinase release over the first 45 min of reperfusion was significantly reduced in hypertrophied hearts treated with cariporide. The endothelium-dependent coronary vasodilation to 5-hydroxytryptamine was observed in all sham-operated hearts before cardiac arrest, however, it was significantly impaired following cardioplegic ischemia and reperfusion. Hypertrophied hearts demonstrated markedly impaired endothelium-dependent and -independent coronary vasodilations during both pre- and post-ischemic period that were not affected by the treatment with cariporide. The cardioprotective effects of the NHE inhibitor cariporide are also manifest in hypertrophied myocardium, which supports the potential usefulness of NHE inhibition in the setting of cardiac surgery.

A Lithium-induced Conformational Change in Serotonin Transporter Alters Cocaine Binding, Ion Conductance, and Reactivity of Cys-109

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.

Protective effect of SM-20550, a selective Na+ - H+ exchange inhibitor, on ischemia-reperfusion-injured hearts.

The protective effects of Na+ - H+ exchange inhibitors SM-20550 (SM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) against ischemia-reperfusion injury were investigated in guinea pig Langendorff hearts. The changes in intracellular pH (pHi), high-energy phosphates, and biologic intracellular active ions ([Na+]i and [Ca2+]i) were regarded using the 31P-NMR and specific fluorescent signals from the heart tissues together with simultaneous recordings of the left ventricular developed pressure (LVDP). The recovery rate of LVDP from ischemia (40 min) by reperfusion was 36.8% in the control experiments, whereas in the presence of SM 10(-7) M, a gradual increase to 75.9% (55.5% with 10(-8) M), in contrast to EIPA (10(-7) M), 47.5% was observed. SM 10(-7) M restored the ATP level by 70% in 40-min reperfusion, which was already higher than the control in the latter half (20-40 min) of the ischemic period. The recovery rate of phosphocreatine by pretreatment of the heart with SM 10(-7) M was 75% in 40 min reperfusion. The pHi estimated from Pi/phosphocreatine chemical shift became highly acidic in ischemic heart so that SM 10(-7) M caused slight but significant pHi reduction from control pHi of 5.89 to 5.75. The level returned to pHi at around 7.38 during 30-40 min reperfusion, and the recovery was significantly greater than the control pHi of 7.24. The fura-2 Ca2+ or SBFI-Na+ signals during Langendorff ischemia heart increased, and rapidly returned to the control level after the reperfusion. SM suppressed the [Na+]i or [Ca2+]i elevation induced in the late stage during ischemia, resulting in LVDP restoration after reperfusion; Diastolic Ca2+ in the end period of ischemia, SM 10(-7) M 194% versus drug-free 220.7%. Na+: SM 10(-7) M 121.6% versus drug-free 128.0%. The present results suggest that the selective Na+ - H+ exchange inhibitor SM is promising as a potent and specific protective agent against ischemia-reperfusion injuries with Ca2+ overload induced via Na+ - H+, Na+ - Ca2+ exchange.

Effects of SM-20550, a selective Na+-H+ exchange inhibitor, on the ion transport of myocardial mitochondria.

The effect of a novel Na+-H+ exchange inhibitor, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate] (SM) on the ion transport of myocardial mitochondria was studied using ion fluorometry and superfusion techniques. Isolated mitochondria from the guinea-pig heart were pre-loaded with fluoroprobes of either BCECF AM for H+, SBFI AM for Na+ or fura-2 AM for Ca2+. Initially, the treated mitochondria were superfused with a normal medium (MOPS-buffer, pH 7.4, 24 degrees C), subsequently fluorometric experiments on the Na+, H+, Ca2+ mobilization across the mitochondrial membrane were performed. The intramitochondrial pH (pHm) was increased by the superfusion of Na+ at physiological cytosolic concentrations of 10 mM, indicating the existence of a Na+-H+ exchange in mitochondrial membranes. The Na+ induced elevation of pH was dose-dependently inhibited by SM 1 microM (delta pHm; 45% as drug-free 100%), and 10 microM (delta pHm; 70%), as observed in our experiments with the myocardial sarcolemmal membrane. The selective Na+-H+ exchange inhibitor SM reduced such pHm elevations more markedly than that of EIPA [5-(N-ethyl-N-isopropyl) amiloride]. The Na+-H+ exchange inhibitors, SM and EIPA suppressed the intramitochondrial Ca2+ elevation ([Ca2+]m) brought on by external Ca2+ concentration changes: The pretreatment with SM 1 microM, 10 microM and EIPA 10 microM reduced the [Ca2+]m influx by 28.3, 56.5 and 63%, respectively. Additionally, the [Ca2+]m elevation induced by acidification of the perfusate was reduced by the prior infusion of SM and EIPA. Pretreatment of mitochondria with SM or EIPA which had beneficial effects on the left ventricular developed pressure (LVDP) in the ischemia-reperfusion injury of Langendorff hearts, reduced the intramitochondrial Na+ and pHm levels, indicating interplay of the inhibitory mechanism of Ca2+-uptake into mitochondria coupled with Na+-H+ exchange. These findings suggested that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due in part to the Ca2+-paradox at the mitochondria level.

A Na +–H + exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion

Na +–H + exchange inhibitors may reduce myocardial damage after reperfusion. However, their effects on microvascular deterioration are not known. We examined the potency of a novel Na +–H + exchange inhibitor, SM-20550 [ N-(Aminoiminomethyl)-1,4-dimethyl-1 H-indole-2-carboxamide methanesulfonate], and its effects on microvascular damage after reperfusion. In an in vitro study, the Na +-H + exchange inhibiting activity of SM-20550 was about 10 times greater than that of ethylisopropyl amiloride. In in vivo experiments, we occluded the left circumflex coronary artery in 29 dogs for 2 h and then reperfused for 5 h. SM-20550 was administered either before ischemia ( n=11) or before reperfusion ( n=7). Another 11 dogs served as controls. We found that SM-20550 not only improved coronary vasodilator responses to acetylcholine and adenosine after reperfusion, but also reduced infarct size ( P<0.01). Intramyocardial bleeding, which should reflect microvascular damage, was not found in dogs with SM-20550 treatment. Infarct size was correlated inversely with collateral blood flow in control (both, P<0.01) but not in SM-20550-treated animals. Furthermore, SM-20550 significantly suppressed ventricular fibrillation during both ischemia and reperfusion. These results suggest that protective effects of Na +–H + exchange inhibitors on reperfused myocardium are due at least in part to microvascular protection.

Effect of verapamil on restoration of cardiac performance in raised [K+]o by adrenergic stimulation in the rabbit.

Modulation of the L-type calcium channel by catecholamines improves action potential parameters in single ventricular myocytes depolarized by high [K+]o Tyrode. Whether this modulation is important in offsetting the negative effects of hyperkalaemia in the whole heart is not known. We tested the effects of the calcium channel antagonist, verapamil, on restoration of cardiac performance by adrenergic stimulation in high [K+]o in anaesthetized rabbits and isolated perfused working rabbit hearts. Raised [K+]o decreased SBP, LVP and LVdP/dtmax in vivo ([K+]a 8.6 +/- 0.2 mM; n = 10) and aortic flow (AF) in the isolated heart (8 mM [K+]o Tyrode; n = 25). However, the negative effects of raised [K+]a were offset by isoprenaline (Iso, 1 microgram kg-1 min-1 i.v.) in vivo and by noradrenaline (NA, 80 nM) in the isolated heart. Verapamil (0.15 mg kg-1 i.v.; 15 nM isolated heart) markedly potentiated the negative inotropic effects of raised [K+]o in both preparations. Verapamil attenuated the effect of isoprenaline in vivo but in the isolated heart, the protective effect of NA in 8 mM [K+] Tyrode (AF 97 +/- 10 mL min-1 in 8 mM [K+]o compared with AF 141 +/- 8.5 mL min-1 in 8 mM [K+]o + NA) was offset by the drug (90 +/- 8 mL min-1 in 8 mM [K+]o + NA + V). Furthermore, verapamil abolished aortic flow in 8 mM [K+]o alone. These findings suggest that the heart may be critically dependent on modulation of intracellular calcium in order to tolerate concentrations of K+ similar to those seen during a short burst of intensive exercise ([K+]a 8.6 mM).

Specific protection by Na+ or Li+ of the F1F0-ATPase of Propionigenium modestum from the reaction with dicyclohexylcarbodiimide

Incubation of the purified F1F0-ATPase of Propionigenium modestum with dicyclohexylcarbodiimide (DCCD) led to inactivation of the enzyme in a strongly pH-dependent manner. Rapid inactivation occurred at pH 5-7, while the increase of the pH from 7 to 9 resulted in a continuous reduction of the inactivation rate. In the presence of Na+ ions, the ATPase was specifically protected from inactivation by DCCD. The protective effect of Na+ was most pronounced at pH 9.0 and less significant at pH 7.0. In addition to Na+, Li+ also protected the ATPase from inactivation by DCCD, but approximately 10 times higher concentrations were required for the same effect. Similarly, the Na+ concentration causing half-maximal stimulation of ATPase activity was about 10 times below the Li+ concentration required for the same activation. It is concluded from these results that a binding site is present for Na+ or Li+ on the enzyme with an about 10 times lower affinity for the latter alkali ion, which when occupied stimulates ATPase activity and protects it from inactivation by DCCD. Inactivation of ATPase activity by DCCD correlated well with a specific labeling of subunit c of the enzyme in the presence of the [14C]DCCD derivative. Like ATPase inactivation, the labeling was promoted by more acidic pH values and inhibited by Na+ ions. We suggest from these data that the DCCD-reactive amino acid residue of subunit c (most likely Glu-65) must be protonated for the reaction with the carbodiimide and provides the Na(+)-binding site in its deprotonated state. Dissociation of the carboxylic acid (at high pH) and binding of Na+ ions to the carboxylate thus abolish the reactivity toward DCCD.

Uptake and Phytotoxicity of the Herbicide Metsulfuron Methyl in Corn Root Tissue in the Presence of the Safener 1,8-Naphthalic Anhydride

Growth of Zea mays L. cv Potro roots was inhibited by the herbicide metsulfuron methyl (MSM) at the lowest concentration tested: 5 nanomoles per liter. Pretreatment of corn seeds with commercial 1,8-naphthalic anhydride (NA) at 1% (w/w) partially reversed MSM-induced root growth inhibition. MSM at a concentration of 52 nanomoles per liter was taken up rapidly by roots and accumulated in the corn tissue to concentrations three times those in the external medium; the safener NA increased MSM uptake up to 48 hours. The protective effect of NA was related to the ability of the safener to increase the metabolism of MSM; tenfold increases in the metabolic rates of MSM were observed in NA-pretreated corn seedlings grown for 48 hours on 52 nanomolar [(14)C]MSM solution. DNA synthesis determined by measurement of [(3)H]thymidine incorporation into DNA was inhibited by root MSM applications; after a 6-hour application period, 13 nanomolar MSM solution reduced DNA synthesis by 64%, and the same reduction was also observed with NA-pretreated seedlings. Pretreatment of corn seeds with safener NA did not increase the acetolactate synthase activity in the roots and did not change, up to 13 micromoles per liter, the in vitro sensitivity of roots to MSM.

Protective effect of Na + and K + against inactivation of (Na + + K +-ATPase by high concentrations of 2-mercaptoethanol at high temperatures

Purified dog kidney (Na + + K +)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50–55°C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the β-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na + + K +)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na + and K + bound to their respective ion-binding sites on the α-subunit exert a protective effect on a disulfide bond on the β-subunit. This suggests some sort of interaction between the α- and the β-subunits.

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: Effect of sodium chloride on bactericidal activity

Bactericidal activity of heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) was found to be inhibited by low molar (10 to 100 m m) concentrations of the monovalent cations Na + and K +. The protective effect of Na + was shown to be species dependent and inversely proportional to the sensitivity of the test microorganism as determined by bactericidal titer. The effect of Na + (100 m m) on bactericidal activity in whole (unheated) LAL, when assayed against a sensitive species ( Escherichia coli), was negligible and in direct contrast to data obtained when using 60°C extracts of LAL. Increasing the concentration of Na + to reflect levels in L. polyphemus blood (400 to 500 m m) afforded only minimal protection for sensitive bacterial species. These data suggested that whole LAL may contain a heat-labile substance (protein?) capable of binding Na +, obviating the inhibitory effect of Na + on bactericidal activity. Further evidence for the existence of this cation-binding substance was obtained when 55°C extracts of LAL were found refractory to Na + concentrations which totally abolished bactericidal activity in 60°C extracts. The mechanism of Na + inhibition of bactericidal activity and the role of the cation-binding substance in L. polyphemus host defense against bacterial invasion and dissemination is discussed.

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K + with Na + effectively prevented the lysis by SCN −. K + salts of NO 3 −, Br −, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN − than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN − is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na + observed here was considered to be manifested by specific interactions of Na + with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.

Thallium inhibition of ouabain-sensitive sodium transport and of the ( [formula omitted])-ATPase in human erythrocytes

The influence of Tl + on Na + transport and on the ATPase activity in human erythrocytes was studied. 0.1–1.0 mM Tl + added to a K +-free medium inhibited the ouabain-sensitive self-exchange of Na + and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5–10 mM external Tl + caused inhibition of the ouabain-sensitive 22Na efflux as well as the ( Na + + Tl + )-ATPase. Competition between the internal Na + and rapidly penetrating thallous ions at the inner Na +-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl +. An increase of the internal Na + concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl +. A protective effect of Na + was also demonstrated on the ( Na + + Tl + )-ATPase of fragmented erythrocyte membranes studied at various Na + and Tl + concentrations.

The effects of cations on the inhibition of K plus-activated phosphatase by beryllium.

The effect of beryllium on K+-activated phosphatase was investigated. Preincubation of the enzyme preparation with BeCl2 in the presence of Mg2+ caused reduction of K+-activated phosphatase activity, while preincubation with BeCl2 only caused no reduction. Addition of K+, Rb+ or NH4+ but not Li+ with Mg2+ and BeCl2 during preincubation increased the inhibition rate. Under the same conditions, Na+ decreased the rate of inhibition. This protective effect of Na+ was greatest in the absence of K+ and decreased with increase in the concentration of K+. The effects of cations on the inhibition of K+-activated phosphatase by beryllium were compared with their effects on that of (Na+ + K+)-activated ATPase [ATP phosphohydrolase, EC 3. 6.1. 3] and results suggested that there is a close linkage between these two enzyme activities. It also seemed that K+ interacted with the enzyme even in the absence ofp-nitrophenyl phosphate and that this interaction was intimately related with the activation of Reactivated phosphatase by K+. Results of kinetic analyses of the activation of Reactivated phosphatase by K+ and the effect of ouabain on the inhibition rate indicated that there are at least two different ways of interaction of K+ with enzyme.