Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Characterization of microsomal ATPases from developing human placenta

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na⁺ + K⁺ + Mg²⁺, Mg²⁺, and Ca²⁺ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg²⁺-dependent and Na⁺ + K⁺ + Mg²⁺-dependent ATPases possess nearly the same K_m (apparent) for ATP, while the Ca²⁺-dependent enzyme shows a different one. Mg²⁺-dependent ATPase shows higher substrate affinity than Ca²⁺-dependent ATPase, although the V_max of the Mg²⁺-dependent enzyme is lower than that of the latter. However, for each enzyme, the K_m remains almost constant and V_max varies during ontogenic development. V_max of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.     

Variable ATPase composition of human tumor plasma membranes

Purified plasma membranes from several transplantable human tumors exhibit very high Mg²⁺-dependent ATPase activities. Three types of Mg²⁺-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na⁺, K⁺-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg²⁺-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na⁺ (or K⁺) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg²⁺-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed.     

Effect of the diazonium salt of sulfanilic acid on human erythrocyte ATPase activity

External treatment of human erythrocytes with the diazonium salt of sulfanilic acid does not inhibit the Mg²⁺-dependent ATPase but does markedly inhibit the Ca²⁺-stimulated ATPase activity. Inhibition of the (Na⁺ + K⁺)-dependent activity is dependent upon the concentration of diazonium salt used. Treatment of membrane fragments does not irreversibly inhibit the (Na⁺ + K⁺)-dependent ATPase even though the diazonium salt binds covalently to membrane components. However, the Mg²⁺-dependent and Ca²⁺-stimulated ATPase activities are irreversibly inhibited. ATP and Mg-ATP will completely protect the (Na⁺ + K⁺)-dependent ATPase when present during treatment of membrane fragments with the diazonium salt, but only Mg-ATP will protect the Mg²⁺-dependent ATPase from inhibition. The Ca²⁺-stimulated ATPase activity is not protected.     

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.     

Quantitative aspects of the interaction between ouabain and (Na + K)-activated ATPase in vitro

The inhibitory effect of ouabain on (Na⁺ + K⁺)-activated ATPase (Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na⁺, Mg²⁺ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na⁺ + K⁺)-activated ATPase reaction was subsequently started by the addition of a KCl solution.     

Microsomal (Na + K)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors

A method is described for the extraction of microsomal ouabain-sensitive (Na⁺ + K⁺)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na⁺ + K⁺)-stimulated activity in the range of 30–40 nmol · mg⁻¹ · min⁻¹ at 26 °C. This portion, which is also ouabain sensitive, is about half of the total activity in media containing Mg²⁺, Na⁺ and K⁺. These preparations also contain Mg²⁺-dependent or Ca²⁺-dependent activities which are not additive and which are not significantly affected by ouabain, Na⁺, K⁺ or Li⁺.The activations of the ouabain-sensitive ATPase activity by Mg²⁺, Na⁺, and K⁺ are similar to those described in other tissues. It is found that Li⁺ does not substitute for Na⁺ as an activator but in high concentrations does produce partial activation in the presence of Na⁺ with no K⁺. These results are pertinent to the reported observations of ouabain-sensitive Li⁺ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li⁺ on the sodium pump.     

Tissue specificity of dopamine effects on brain ATPases

Dopamine inhibits Mg²⁺,Na⁺,K⁺- and Na⁺,K⁺-ATPase activities but does not modify Mg²⁺-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na⁺,K⁺-, and Mg²⁺-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. These results indicate tissue specificity of dopamine effects with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.     

Effect of corpus cardiacum extracts on the ATPase activity of locust rectum

The Mg²⁺ dependent and Na⁺K⁺-activated ATPase activities of microsomal preparations from the rectum of Locusta migratoria were both stimulated, to varying extents, by crude extracts of the corpora cardiaca of this species. Mg²⁺ ATPase activity increased by approximately 549% whereas the hormonal stimulation of Na⁺K⁺-activated ATPase depended upon the concentration of sodium and potassium ions. At 100 mM Na⁺ and 20 mM K⁺, conditions which approximate to optimum for this enzyme system, Na⁺K⁺-activated ATPase activity increased by about 14%. At sub-optimum concentrations of these ions, i.e. 50 and 5 mM Na⁺ and K⁺ respectively, the increase in Na⁺K⁺-activated ATPase activity was about 205%. Ouabain at a concentration of 10^?3 M completely abolished this stimulated activity and was consistently effective in partially reducing the stimulation of Mg²⁺ ATPase activity by corpora cardiaca extracts.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.     

Lead ion activates phosphorylation of electroplax Na+- and K+-dependent adenosine triphosphatase ((NaK)-ATPase) in the absence of sodium ion.

PbCl₂ in micromolar concentrations stimulates phosphorylation of electroplax microsomal protein in the absence of Na⁺. Other divalent cations showed little or no such effect. The (Mg²⁺ + Pb²⁺)- and (Mg²⁺ + Na⁺)-dependent membrane-bound protein kinase activities in electroplax particulate preparations exhibit properties in common, including their acid stability, ouabain sensitivity, ATP specificity, and molecular size. It is concluded that the (Mg²⁺ + Pb²⁺)-dependent phosphoprotein is part of the Na⁺-, K⁺-dependent adenosine triphosphatase [(NaK)ATPase]. The Pb²⁺-dependent product, in contrast to the Na⁺-dependent one, is insensitive to K⁺ and the hydrolysis of ATP is thus inhibited.     

Properties of (Na+ + K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124–132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na⁺ + K⁺)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na⁺ + K⁺)-activated ATPase activity was documented by demonstrating specific cation requirements for Na⁺ and K⁺, while the divalent cation, Ca²⁺, and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na⁺ + K⁺)-activated ATPase activity averaged 10.07 ± 2.80 μmol P_i/mg protei per h compared to 50.03 ± 11.41 for Mg²⁺-activated ATPase and 58.66 ± 10.07 for 5′-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na⁺ + K⁺)-activated ATPase without any effect on Mg²⁺-activated ATPase. Both (Na⁺ + K⁺)-activated ATPase and Mg²⁺-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na⁺ + K⁺)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na⁺ + K⁺)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Transport ATPase: further studies on the properties of the thimerosal-treated enzyme.

Our previous studies showed that when ethylmercurithiosalicylate (thimerosal) interacts with the transport ATPase of the guinea pig kidney under specified conditions, the Na⁺ + K⁺-dependent ATPase activity is inhibited, while the Na⁺-dependent ATPase, the Na⁺ + ATP-dependent phosphorylation of the enzyme, and the K⁺-dependent discharge of the phosphoenzyme seem to be unaffected. Here we describe other properties of the thimerosal-treated enzyme: Na⁺-dependent ADP-ATP exchange, Na⁺-dependent UTPase, and K⁺-dependent p-nitrophenylphosphatase activities of the modified enzyme are not inhibited. Kinetics of the Na⁺ effect on the UTPase activities of the native and the modified enzyme are the same. However, K⁺ has a greater inhibitory effect on the Na⁺-UTPase of the modified enzyme than on the Na⁺-UTPase of the native enzyme. The increase in the apparent affinity of the thimerosal-treated enzyme for K⁺ is also evident from the kinetics of the K⁺ effect on p-nitrophenylphosphatase. Neither the native enzyme nor the modified enzyme catalyzes a P₁-ATP exchange. The uninhibited activities of the thimerosal-treated enzyme are sensitive to ouabain. These data provide further support for those reaction mechanisms in which the existence of two ATP sites within the enzyme is assumed.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Partial characterization of an endogenous factor which modulates the effect of catecholamines on synaptosomal Na+, K+-ATPase

We have previously presented evidence for the existence of a brain soluble factor which mediates the stimulation of synaptosomal ATPases by catecholamines. The stimulation of synaptosomal ATPases by dopamine plus brain soluble fraction was not modified if the soluble fraction was heated for 5 min at 95°C. One day after preparation, the soluble factor inhibited the Na⁺, K⁺-ATPase, but not the Mg²⁺-ATPase activity, and subsequent addition of noradrenaline stimulated the ATPases activities. The inhibitory effect of a 24 h soluble fraction disappeared if the soluble fraction was dialyzed; in this case, noradrenaline did not activate the enzyme activities. Gel filtration in Sephadex G-50 permitted separating a subfraction which inhibited ATPase activity (peak II) from another which stimulated ATPase activity (peak I). Peak I stimulated both Na⁺, K⁺, and Mg²⁺ ATPases. Peak II inhibited only Na⁺, K⁺-ATPase, and when stored acidified, it mediated ATPases stimulation by noradrenaline.Special Issue dedicated to Prof. Eduardo De Robertis.     

Effects of Na+ and/or K+ on the Mg2+-dependent ATPase activities in shrimp (Macrobrachium amazonicum) gill homogenates

• 1.1. Homogenates of gills from the freshwater shrimp M. amazonicum exhibit the following ATPase activities: (i) a basal, Mg²⁺-dependent ATPase; (ii) an ouabain-sensitive, Na⁺ + K⁺-stimulated ATPase; (iii) an ouabain-insensitive, Na⁺-stimulated ATPase; and (iv) an ouabain-insensitive, K⁺-stimulated ATPase.
• 2.2. K⁺ suppresses the Na⁺-stimulated ATPase activity in a mixed-type kind of inhibition, whereas Na⁺ does not exert any noticeable effect on the K⁺-stimulated ATPase activity.
• 3.3. The Na⁺- and the K⁺-stimulated ATPase activities are totally inhibited by 5 mM ethacrynic acid in the incubation medium.
• 4.4. The Na⁺- and the K⁺-stimulated ATPase activities are not expressions of the activation of a Ca-ATPase.
• 5.5. The possible localization and roles of the described ATPases within the gill epithelium are briefly discussed and evaluated.

     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.