Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Thiophosphoryl guanine nucleotide analogues inhibit the (Na+,K+)-ATPase.

The effects of several guanine nucleotide analogues on (Na+,K+)-ATPase activity of membranes isolated from several tissues were analyzed to determine if a G-protein might be involved in the hormonal regulation of the (Na+,K+)-ATPase. Submillimolar concentrations of GTP gamma S, but not GMPPNP, inhibit rat skeletal muscle and axolemma, but not kidney, (Na+,K+)-ATPase activity. Furthermore, GDP beta S does not reverse GTP gamma S inhibition, but rather itself slightly inhibits (Na+,K+)-ATPase activity. Dithiothreitol can block and reverse GTP gamma S inhibition of skeletal muscle (Na+,K+)-ATPase; the results obtained with axolemma membranes are complicated by the inhibition of (Na+,K+)-ATPase activity in these membranes by DTT. Results showing that high membrane concentrations can mute the inhibitory action of GTP gamma S suggest that a minor contaminant in GTP gamma S preparations is responsible for inhibiting (Na+,K+)-ATPase activity. Neither vanadate, a heavy metal, GDP, phosphate, nor thiophosphate, however, is responsible for this inhibition, and the inhibitory activity elutes with GTP gamma S from Sephadex G-10 columns. It is concluded that GTP gamma S or a structural derivative of GTP gamma S inhibits the (Na+,K+)-ATPase, in a tissue-specific manner, not by interaction with a G-protein as a GTP analogue, but through a direct chemical interaction with the (Na+,K+)-ATPase or some regulatory protein. The terminal SH group of the nucleotide analogue is probably required for this interaction.     

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.     

Inhibition of rat cardiac and renal Na+,K+-ATPase by high sodium concentrations and vanadate

In the present study, rat renal Na+,K+-ATPase was found to be more sensitive to inhibition by high Na+ concentrations (100-400 mM) than was rat cardiac Na+,K+-ATPase. K+ was more effective in reversing the inhibition by Na+, of cardiac relative to renal Na+,K+-ATPase. Rat renal Na+,K+-ATPase was also more sensitive than cardiac Na+,K+-ATPase to inhibition by vanadate over this range of Na+ concentrations. These results support the hypothesis that vanadate may selectively regulate Na+,K+-ATPase in the kidney, and they may also help explain the natriuretic and diuretic effects of vanadate in rats. Inhibition of renal Na+,K+ATPase by Na+, may also help explain, in part, the natriuretic and diuretic effects of acute saline loading.     

Dog kidney (Na+, K+)-ATPase is more sensitive to inhibition by vanadate than human red cell (Na+, K+)-ATPase

(Na⁺, K⁺)-ATPase (EC 3.6.1.3) from kidney is more sensitive to inhibition by vanadate than red cell (Na⁺,K⁺)-ATPase. The difference appears to be in the apparent affinities of the two enzymes for K⁺ and Na⁺ at sites where K⁺ promotes and Na⁺ opposes vanadate binding. As a result of Na⁺-K⁺ competition at these sites, reversal of vanadate inhibition was accomplished at lower Na⁺ concentrations in red cell than in kidney (Na⁺,K⁺)-ATPase. It is possible that vanadate could selectively regulate Na⁺ transport in the kidney.     

Immunochemical characterization of a functional site of (Na+,K+)-ATPase

Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vanadate sensitivity of Na+, K+-ATPase from Schistosoma mansoni and its modulation by Na+, K+ and Mg2+

Vanadate inhibitory effects on Na+, K+-ATPases from carcass of Schistosoma mansoni and from lamb kidney outer medulla were compared in the presence of various concentrations of Na+, K+ and Mg2+. Depending on the ionic conditions, the schistosomal Na+, K+-ATPase was 2.4- to 175-fold less sensitive to vanadate than the lamb kidney enzyme. In 100 mM Na+, 3 mM K+ and 3 mM Mg2+, schistosomal Na+, K+-ATPase was surprisingly resistant to vanadate (I50 = 944 microM). The difference in vanadate sensitivity between schistosomal and lamb Na+, K+-ATPases may be due to a species difference in the efficacy of Na+, K+ and Mg2+ in promoting conformational changes between E1 and E2 forms of the enzyme.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 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. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 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.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

Immunochemical evidence for a transmembrane orientation of both the (Na+, K+)-ATPase subunits

Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Studies on ouabain-complexed (Na+ +K+)-ATPase carried out with vanadate

Vanadate is able to promote the binding of ouabain to (Na+ +K+)-ATPase and it is shown that vanadate is trapped in the enzyme-ouabain complex. Also ouabain-bound enzyme, the formation of which was facilitated by (Mg2+ +Na+ +ATP) or (Mg2+ +Pi), is accessible to vanadate when washed free of competing ligands used for the promotion of ouabain binding. For vanadate binding to (Na+ +K+)-ATPase and to enzyme-ouabain complexes a divalent cation (Mg2+ or Mn2+) is indispensable, indicating that the cation does not remain attached to the ouabain-bound enzyme. K+ further increases vanadate binding in the absence of ouabain, but seems to have no additional role in case of vanadate binding to enzyme-ouabain complexes. Mn2+ is more efficient than Mg2+ in promoting binding of vanadate and ouabain to (Na+ +K+)-ATPase. That K+ in combination with Mn2+, in analogy with the effect in combination with Mg2+, increases the equilibrium binding level of vanadate and decreases that of ouabain does not seem to favour the hypothesis of selection of a special E2-subconformation by Mn2+. The vanadate-trapped enzyme-ouabain complex was examined for simultaneous nucleotide binding which could demonstrate a two-substrate mechanism per functional unit of the enzyme. The acceleration by (Na+ +ATP) of ouabain release from the (Mg2+ +Pi)-facilitated enzyme-ouabain complex does not, as anticipated, support such a mechanism. On the other hand, the deceleration of vanadate release as well as of ouabain release from a (Mg2+ +vanadate)-promoted complex could be consistent with a two-substrate mechanism working out-of-phase.     

Antigenic differences in (Na+, K+)-ATPase preparations isolated from various organs and species.

Antisera to purified (Na+, K+)-ATPase raised in rabbits and in sheep were purified by an absorption procedure employing purified canine kidney (Na+, K+)-ATPase. The antibodies were fractionated into two components, one which inhibited catalytic activity, and a second which inhibited ouabain binding. Under certain conditions, the fraction that inhibited ouabain binding also inhibited catalytic activity, and the effectiveness of both was dependent to some extent on the ligands present in the incubation medium. Thus, both antibody fractions appeared to detect conformations of the enzyme that depended upon ligand-induced perturbations. When the antibody raised against catalytic activity was incubated with erythrocyte membrane fragments, an inhibition of the (Na+, K+)-ATPase occurred, but only minimal or no effect on potassium influx or on digoxin-induced inhibition of potassium flux in intact erythrocytes was noted. In a similar experiment, however, the antibody against ouabain binding significantly inhibited potassium influx, suggesting specificity in terms of the macromolecular surfaces of the pump which were exposed to the external medium. We concluded that there may be organ and species differences among (Na+, K+)-ATPase preparations. Antibodies prepared in rabbits and sheep were fractionated by absorption to dog brain enzyme. Both the antibody fraction which bound to the brain enzyme and that which did not bind inhibited the dog kidney (Na+, K+)-ATPase, but only the former inhibited dog brain (Na+, K+)-ATPase. When the two fractions were recombined, inhibition was restored to the extent of the unfractionated antibody.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney

The (Na+,K+)-ATPase ATP hydrolyzing activity from rabbit kidney medulla basolateral membrane vesicles was studied as a function of the cholesterol content of the basolateral membranes. The cholesterol content of the membranes was modified by incubation with phospholipid vesicles. When the cholesterol content was increased above that found in the native membrane, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. When the cholesterol content was decreased from that found in the native membranes, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. Analogous effects were found with the K+-activated phosphatase activity of the same membrane vesicles. Therefore, at low cholesterol contents, cholesterol was stimulatory, and at high cholesterol contents, cholesterol was inhibitory. The structural specificity of this effect was tested by introducing lanosterol and ergosterol as 50% of the membrane sterol. Ergosterol was the least effective at supporting (Na+,K+)-ATPase ATP hydrolyzing activity, while lanosterol was more effective, but still not as effective as cholesterol.     

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.     

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.     

Crystallization patterns of membrane-bound (Na+ +K+)-ATPase

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump.