Interaction of some Pd(II) complexes with Na+ / K+-ATPase: inhibition, kinetics, prevention and recovery

The aim of this work was to investigate the influence of [PdCl4]2-, [PdCl(dien)]+ and [PdCl(Me4dien)]+ complexes on Na+ / K+-ATPase activity. The dose-dependent inhibition curves were obtained in all cases. IC50 values determined by Hill analysis were 2.25 x 10(-5) M, 1.21 x 10(-4) M and 2.36 x 10(-4) M, respectively. Na+ / K+-ATPase exhibited typical Michelis-Menten kinetics in the presence of Pd(II) complexes. Kinetic parameters (Vmax, Km) derived using Eadie-Hofstee transformation indicated a noncompetitive type of Na+ / K+-ATPase inhibition. The inhibitor constants (Ki) were determined from Dixon plots. The order of complex affinity for binding with Na+ / K+-ATPase, deducted from Ki values, was [PdCl4]2- > [PdCl(dien)]+ > [PdCl(Me4dien)]+. The results indicated that the potency of Pd(II) complexes to inhibit Na+/ K +-ATPase activity depended strongly on ligands of the related compound. Furthermore, the ability of SH-donor ligands, L-cysteine and glutathione, to prevent and recover the Pd(II) complexes-induced inhibition of Na+ / K+-ATPase was examined. The addition of 1 mM L-cysteine or glutathione to the reaction mixture before exposure to Pd(II) complexes prevented the inhibition by increasing the IC50 values by one order of magnitude. Moreover, the inhibited enzymatic activity was recovered by addition of SH-donor ligands in a concentration-dependent manner.     

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.     

Effects of digoxin and gitoxin on the enzymatic activity and kinetic parameters of Na+/K+-ATPase

Inhibition of Na+/K+-ATPase activity from human erythrocyte membranes and commercial porcine cerebral cortex by in vitro single and simultaneous exposure to digoxin and gitoxin was investigated to elucidate the difference in the mechanism of the enzyme inhibition by structurally different cardiac glycosides. The drugs exerted a biphasic dose-dependent inhibitory effect on the enzyme activity in both tissues, supporting the existence of two sensitive Na+/K+-ATPase isoforms. The IC50 values for the low and high affinity isoforms were calculated from the inhibition curves using mathematical analysis. The Hill coefficient (n) fulfilled the relationship 1 < n < 3, suggesting cooperative binding of inhibitors to the enzyme. Kinetic analysis showed that digoxin and gitoxin inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction. Both the isoforms were always more sensitive to gitoxin. The erythrocyte enzyme was more sensitive to the inhibitors in the range of low concentrations but the commercial cerebral cortex enzyme exerted a higher sensitivity in high inhibitors affinity concentration range. By simultaneous exposure of the enzyme to digoxin and gitoxin in combinations a synergistic effect was achieved by low inhibitor concentrations. An antagonistic effect was obtained with erythrocyte membrane enzyme at high inhibitors concentration.     

Effects of glycyrrhizin and glycyrrhetinic acid on (Na+ + K+)-ATPase of renal basolateral membranes in vitro

Studies were made on the direct effects of glycyrrhizin and its aglycone, glycyrrhetinic acid on the activities of (Na+ + K+)-ATPase and (Ca2+ + Mg2+)-ATPase, a membrane bound Na+ and Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney. Glycyrrhetinic acid inhibited the activity of the Na+-pump enzyme dose-dependently (IC50 = 1.5 x 10(-4) M), but had no effect on that of the Ca2+-pump enzyme of kidney BLM and homogenates. Glycyrrhizin also inhibited the Na+-pump enzyme activity but had less effect (IC50 = 2 x 10(-3) M). The effects of these compounds were due to competitive inhibition with ATP binding to the enzyme (Ki = 12 microM) and so were different from that of ouabain, which inhibits the Na+-pump by binding to its extracellular K+-binding site. The direct effect of glycyrrhetinic acid on the membrane may be important role in the multiple actions of licorice.     

Interaction of palytoxin and cardiac glycosides on erythrocyte membrane and (Na+ + K+) ATPase

Palytoxin (PTX), at extremely low concentrations (0.01-1 nM), caused K+ release from rabbit erythrocytes. Among the various chemical compounds tested, cardiac glycosides potently inhibited the PTX-induced K+ release. The order of inhibitory potency (IC50) was cymarin (0.42 microM) greater than convallatoxin (0.9 microM) greater than ouabain (2.3 microM) greater than digitoxin (88 microM) greater than digoxin (90 microM). Their corresponding aglycones, even at 10 microM, did not inhibit the K+ release, but competitively antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex in narrow concentration ranges (IC50 = 0.15-2.4 microM), suggesting that the inhibition of K+ release is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity, and the sugar moiety of cardiac glycosides is involved in the inhibition. On the other hand PTX, at higher concentrations (greater than 0.1 microM), inhibited the (Na+ + K+)-ATPase activity. However, this inhibitory effect of PTX was not antagonized by ouabain. It is suggested that, compared with ouabain, PTX has additional binding site(s) on the (Na+ + K+)-ATPase.     

Modulation of benthiocarb in vitro inhibited neonate rat (Wistar strain) brain Na+K+-ATPase by norepinephrine

Effect in vitro of benthiocarb, an organocarbamate herbicide on neonate rat (3 day old) brain was studied to understand the interaction of benthiocarb with Na+K+-ATPase. Na+K+-ATPase of the developing rat brain was selected as an index enzyme since alterations in the Na+K+-ATPase activity leads to neuronal dysfunction. The assay of Na+K+-ATPase in the presence of 1-8 mu moles of benthiocarb showed decreased activity and a concentration dependent inhibition of Na+K+-ATPase was noticed upto 7 mu moles of benthiocarb. Based on IC50 values (median concentration), 50% inhibition of the enzyme was observed with 5 mu moles of benthiocarb. Norepinephrine (NE) was selected to study the modulation of benthiocarb inhibited enzyme. Maximum increase (76.7%) of Na+K+-ATPase was noticed with 35 mu moles of NE and effective concentration (EC50) of NE which produced 50% activation of the enzyme was found to be 20 mu moles. This study suggests that NE acts as a protective agent in reversing the benthiocarb in vitro inhibited neonate rat brain Na+K+-ATPase.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

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 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 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 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 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.     

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.     

Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10?nM Ang II for 5?min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30?mM NaCl and 3?mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150?mM NaCl and 3?mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.     

Inhibition of cardiac Na+, K+-ATPase activity by dynorphin-A and ethylketocyclazocine

The effect of various opioids on Na+, K+ -ATPase partially purified from rat heart was examined. Dynorphin-A (1-13), dynorphin-A (1-17) and ethylketocyclazocine (EKC), which are k-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC50 values were 12 microM, 21 microM and 0.38 mM, respectively. Morphine (mu-type agonist), methionine- and leucine-enkephalin (delta-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A (1-13) and EKC was not antagonized by naloxone. Dynorphin-A (1-13) mainly decreased Vmax value without the change of Km value in the activation of Na+, K+-ATPase by ATP, Na+ and K+. Dynorphin-A(1-13) inhibited the partial reactions of Na+, K+-ATPase at the different degree of the potency; the inhibition of K+-stimulated phosphatase was greater than that of Na+-dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na+, K+-ATPase in the heart.     

Properties of Na+, K+-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.     

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.     

Brain Na+, K+-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na(+), K(+)-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075x10(-9) M, 0.58x10(-6) M and 0.97x10(-3) M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80x10(-9) M, 0.56x10(-6) M and 0.21x10(-3) M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na(+), K(+)-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1x10(-9) M, or 5x10(-6) M ouabain, whereas only a trend was recorded in the presence of 1x10(-3) M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na(+), K(+)-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.