Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Two different modes of inhibition of the rat brain Na+,K+-ATPase by triterpene glycosides,psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na⁺,K⁺-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na⁺,K⁺-ATPase with I₅₀ values of 1×10⁻⁴ M and 3×10⁻⁴ M, respectively. PsA significantly stimulated [³H]ATP binding to Na⁺,K⁺-ATPase, weakly increased [³H]ouabain binding to the enzyme, and inhibited K⁺-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [³H]ATP binding to Na⁺,K⁺-ATPase, and had no effect on [³H]ouabain binding to the enzyme. K⁺-Phosphatase activity was inhibited by PsB in parallel with Na⁺,K⁺-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na⁺,K⁺-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney

The rates of association of [³H]ouabain to Na⁺,K⁺-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na⁺,K⁺-ATPase being the fastest. Although the major determinant of the affinity of the Na⁺,K⁺-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na⁺,K⁺-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I₅₀ (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na⁺,K⁺-ATPase activity, approached the value calculated from [³H]ouabain binding. The ratio of the I₅₀ value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na⁺,K⁺-ATPase is not the reason for the faster dissociation rate of this enzyme.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Regulation of Human and Pig Renal Na+,K+-ATPase Activity by Tyrosine Phosphorylation of Their α1-Subunits

Modulation of the physiologically influential Na⁺,K⁺-ATPase is a complex process involving a wide variety of factors. To determine the possible effects of the protein tyrosine phosphatase (PTP) inhibitors dephostatin and Et-3,4-dephostatin on human and pig, renal cells and enzymatic extracts, we treated our samples (15 min–24 h) with those PTP inhibitors (0–100 μM). PTP inhibitors were found to possess a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in both human and pig samples. The inhibition was similarly demonstrated on all cellular, microsomal fraction and purified Na⁺,K⁺-ATPase levels. Despite rigorous activity recovery attempts, the PTP inhibitors’ effects were sustained on Na⁺,K⁺-ATPase activity. Western blotting experiments revealed the expression of both α₁- and β₁-subunits in both human and pig tissues. α₁-Subunits possessed higher tyrosine phosphorylation levels with higher concentrations of PTP inhibitors. Meanwhile, serine/threonine residues of both α₁- and β₁-subunits demonstrated diminished phosphorylation levels upon dephostatin treatment. Accordingly, we provide evidence that Na⁺,K⁺-ATPase can be regulated through tyrosine phosphorylation of primarily their α₁-subunits, using PTP inhibitors.     

Purification,characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol

A protein isolated from goat testis cytosol is found to inhibit Na⁺,K⁺-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I₅₀) occurs is in the nanomolar range. The inhibitor seems to bind Na⁺,K⁺-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na⁺,K⁺-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na⁺,K⁺-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.     

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

Dopamine Oxidation Products Inhibit Na+, K+-ATPase Activity in Crude Synaptosomal–mitochondrial Fraction from Rat Brain

The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50–200?μM) causes dose-dependent inhibition of Na⁺, K⁺-ATPase activity of rat brain crude synaptosomal–mitochondrial fraction during in vitro incubation up to 2?h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na⁺, K⁺-ATPase. Under similar conditions of incubation, DA (200?μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal–mitochondrial proteins and the phenomenon is also prevented by glutathione (5?mM) or cysteine (5?mM).

The available data imply that the inactivation of Na⁺, K⁺-ATPase in this system involves both H²O² and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na⁺, K⁺-ATPase from DA-mediated damage. The inactivation of neuronal Na⁺, K⁺-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease.     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Effects of prostaglandin A2 on Na+, K+-ATPase activity in basolateral plasma membrane of rat intestine in vitro

Prostagladin A₂, which prevents intestinal ulcers produced by administration of nonsteroidal antiinflammatory compounds such as indomethacin, inhibited the Na⁺,K⁺-ATPase activity in basolateral plasma membrane of rat intestine significantly. Prostaglandin A₂ inhibited mainly the Na⁺-dependent phosphorylation step in the overall reaction of Na⁺,K⁺-ATPase. This decrease of the Na⁺,K⁺-ATPase activity by prostaglandin A₂ was due to the decrease of Vmax of the enzyme and of the affinity of the enzyme for Na⁺. It was also suggested that the presence of both Δ^5,6 and Δ^10,11 structure of prostaglandin A₂ may be necessary for the inhibition of the Na⁺,K⁺-ATPase activity.     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

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 doseand 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 ⁸⁶Rb⁺ 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.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.     

Inhibition of membrane transport ATPases by halenaquinol,a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata

Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na⁺, K⁺-ATPases and the rabbit muscle sarcoplasmic reticulum Ca²⁺-ATPase with I₅₀ values of 7.0×10⁻⁷, 1.3×10⁻⁶ and 2.5×10⁻⁶ M, respectively. Halenaquinol also inhibited K⁺-phosphatase activity of the rat brain cortex Na⁺, K⁺-ATPase with an I₅₀ value of 3×10⁻⁶ M. Ouabain-insensitive Mg²⁺-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents

Tricyclohexylhydroxytin, commonly known as Plictran® inhibited Na⁺, K⁺ -ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 μM. Both K⁺ -stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 μM, respectively. Altered pH and Na⁺, K⁺ -ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na⁺, K⁺ -ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na⁺, K⁺ -ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K⁺ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na⁺, K⁺ -ATPase, K⁺ -para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na⁺, K⁺ -ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.