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.     

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+.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. I. Substrate identity

A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor.     

Kinetics of (Na+ + K+)-ATPase: analysis of the influence of Na+ and K+ by steady-state kinetics

The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.     

Ethylenediamine as active site probe for Na+/K+-ATPase

(1) Ethylenediamine is an inhibitor of Na+- and K+-activated processes of Na+/K+-ATPase, i.e. the overall Na+/K+-ATPase activity, Na+-activated ATPase and K+-activated phosphatase activity, the Na+-activated phosphorylation and the Na+-free (amino-buffer associated) phosphorylation. (2) The I50 values (I50 is the concentration of inhibitor that half-maximally inhibits) increase with the concentration of the activating cations and the half-maximally activating cation concentrations (Km values) increase with the inhibitor concentration. (3) Ethylenediamine is competitive with Na+ in Na+-activated phosphorylation and with the amino-buffer (triallylamine) in Na+-free phosphorylation. Significant, though probably indirect, effects can also be noted on the affinity for Mg2+ and ATP, but these cannot account for the inhibition. (4) Inhibition parallels the dual protonated or positively charged ethylenediamine concentration (charge distance 3.7 A). (5) Direct investigation of interaction with activating cations (Na+, K+, Mg+, triallylamine) has been made via binding studies. All these cations drive ethylenediamine from the enzyme, but K+ and Mg+ with the highest efficiency and specificity. Ethylenediamine binding is ouabain-insensitive, however. (6) Ethylenediamine neither inhibits the transition to the phosphorylation enzyme conformation, nor does it affect the rate of dephosphorylation. Hence, we provisionally conclude that ethylenediamine inhibits the phosphoryl transfer between the ATP binding and phosphorylation site through occupation of cation activation sites, which are 3-4 A apart.     

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.     

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.     

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.     

Potassium binding to the (Na+ + K+)-ATPase

The number of K+ bound to the (Na+ + K+)-ATPase has been measured under equilibrium conditions by a differential-titration technique (Hastings, D.F. (1977) Anal. Biochem. 83, 416-432). 5.1 K+ were bound per 32P-labelling site. The K'D for K+ was dependent on the concentration of choline, which was included to give ionic strength. K'D was 59 +/- 2.5 microM with 97 mM choline, 26 +/-1.9 microM with 30 mM choline. The K+ : choline selectivity was 2564 : 1 and the calculated K'D for K+ with zero choline was 11 microM and for choline with zero K+ was 28 mM. 20 microM ATP in the presence of 97 mM choline incresed the K'D for potassium 3-fold to 177 +/- 14 microM. The K'D for K+ with 3 mM Na+ in the presence of 27 mM choline was 81 +/- 10 microM and with 30 mM Na+ without choline 700 +/- 250 microM. The calculated K'D for Na+ at zero K+ and zero choline was 0.6 +/- 0.2 mM. The K+ : Na+ selectivity was 54 : 1.     

Some total and partial reactions of Na+/K+-ATPase using ATP and acetyl phosphate as a substrate

Acetyl phosphate, as a substrate of (Na+ + K+)-ATPase, was further characterized by comparing its effects with those of ATP on some total and partial reactions carried out by the enzyme. In the absence of Mg2+ acetyl phosphate could not induce disocclusion (release) of Rb+ from E2(Rb); nor did it affect the acceleration of Rb+ release by non-limiting concentrations of ADP. In K+-free solutions and at pH 7.4 sodium ions were essential for ATP hydrolysis by (Na+ + K+)-ATPase; when acetyl phosphate was the substrate a hydrolysis (inhibited by ouabain) was observed in the presence and absence of Na+. In liposomes with (Na+ + K+)-ATPase incorporated and exposed to extravesicular (intracellular) Na+, acetyl phosphate could sustain a ouabain-sensitive Rb+ efflux; the levels of that flux were similar to those obtained with micromolar concentrations of ATP. When the liposomes were incubated in the absence of extravesicular Na+ a ouabain-sensitive Rb+ efflux could not be detected with either substrate. Native (Na+ + K+)-ATPase was phosphorylated at 0 degrees C in the presence of NaCl (50 mM for ATP and 10 mM for acetyl phosphate); after phosphorylation had been stopped by simultaneous addition of excess trans-1,2-diaminocyclohexane-N,N,N',N' tetraacetic acid and 1 M NaCl net synthesis of ATP by addition of ADP was obtained with both phosphoenzymes. The present results show that acetyl phosphate can fuel the overall cycle of cation translocation by (Na+ + K+)-ATPase acting only at the catalytic substrate site; this takes place via the formation of phosphorylated intermediates which can lead to ATP synthesis in a way which is indistinguishable from that obtained with ATP.     

The Na+/K+-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.     

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.     

Effects of choline on Na(+)- and K(+)-interactions with the Na+/K(+)-ATPase.

Choline chloride, 100 mM, stimulates Na+/K(+)-ATPase activity of a purified dog kidney enzyme preparation when Na+ is suboptimal (9 mM Na+ and 10 mM K+) and inhibits when K+ is suboptimal (90 mM Na+ and 1 mM K+), but has a negligible effect at optimal concentrations of both (90 mM Na+ and 10 mM K+). Stimulation occurs at low Na+ to K+ ratios, but not at those same ratios when the actual Na+ concentration is high (90 mM). Stimulation decreases or disappears when incubation pH or temperature is increased or when Li+ is substituted for K+ or Rb+. Choline+ also reduces the Km for MgATP at the low ratio of Na+ to K+ but not at the optimal ratio. In the absence of K+, however, choline+ does not stimulate at low Na+ concentrations: either in the Na(+)-ATPase reaction or in the E1 to E2P conformational transition. Together, these observations indicate that choline+ accelerates the rate-limiting step in the Na+/K(+)-ATPase reaction cycle, K(+)-deocclusion; consequently, optimal Na+ concentrations reflect Na+ accelerating that step also. Thus, the observed K0.5 for Na+ includes high-affinity activation of enzyme phosphorylation and low-affinity acceleration of K(+)-deocclusion. Inhibition of Na+/K(+)-ATPase and K(+)-nitrophenylphosphatase reactions by choline+ increases as the K(+)-concentration is decreased; the competition between choline+ and K+ may represent a similar antagonism between conformations selected by choline+ and by K+.     

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.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.     

Cation sidedness in the phosphorylation step of Na+/K+-ATPase

Na+/K+ -ATPase, reconstituted into phospholipid vesicles, has been used to study the localisation of binding sites of ligands involved in the phosphorylation reaction. Inside-out oriented Na+/K+ -ATPase molecules are the only population in this system, which can be phosphorylated, as the rightside-out oriented as well as the non-incorporated enzyme molecules are inhibited by ouabain. In addition, the right-side-out oriented Na+/K+ -ATPase molecules have their ATP binding site intravesicularly and are thus not accessible to substrate added to the extravesicular medium. Functional binding sites for the following ligands have been demonstrated: (i) Potassium, acting at the extracellular side with high affinity (stimulating the dephosphorylation rate of the E2P conformation) and low affinity (inducing the non-phosphorylating E2K complex). (ii) Potassium, acting at the cytoplasmic side with both high and low affinity. The latter sites are also responsible for the formation of an E2K complex and complete with Na+ for its binding sites. (iii) Sodium at the cytoplasmic side responsible for stimulation of the phosphorylation reaction. (iv) Sodium (and amine buffers) at the extracellular side enhancing the phosphorylation level of Na+/K+ -ATPase where choline chloride has no effect. (v) Magnesium at the cytoplasmic side, stimulating the phosphorylation reaction and inhibiting it above optimal concentrations.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: rapid solution exchange with a new technique.

Adsorption of Na+/K+-ATPase containing membrane fragments from pig kidney to lipid membranes allows the detection of electrogenic events during the Na+/K+-ATPase reaction cycle with high sensitivity and time resolution. High stability preparations can be obtained using solid supported membranes (SSM) as carrier electrodes for the membrane fragments. The SSMs are prepared using an alkanethiol monolayer covalently linked to a gold surface on a glass substrate. The hydrophobic surface is covered with a lipid monolayer (SAM, self-assembled monolayer) to obtain a double layer system having electrical properties similar to those of unsupported bilayer membranes (BLM). As we have previously shown (, Biophys. J. 64:384-391), the Na+/K+-ATPase on a SSM can be activated by photolytic release of ATP from caged ATP. In this publication we show the first results of a new technique which allows rapid solution exchange at the membrane surface making use of the high mechanical stability of SSM preparations. Especially for substrates, which are not available as a caged substance-such as Na+ and K+-this technique is shown to be capable of yielding new results. The Na+/K+-ATPase was activated by rapid concentration jumps of ATP and Na+ (in the presence of ATP). A time resolution of up to 10 ms was obtained in these experiments. The aim of this paper is to present the new technique together with the first results obtained from the investigation of the Na+/K+-ATPase. A comparison with data taken from the literature shows considerable agreement with our experiments.     

Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K(+)-ATPase engaged in uncoupled Na(+)-efflux.

In liposomes with reconstituted shark Na+/K(+)-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K(+)-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K+cyt is found to be present. The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of KS1, KS2, KS3 = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K+cyt sites with equal affinity for Ki = 40 mM, similar to KS1 for the first Na+cyt site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperatively in the successive Na+ bindings. pH is found to influence the pattern of K+cyt inhibition: A lowering of the pH potentiates the K+cyt inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.     

Stimulation of p-nitrophenylphosphatase activity of Na+/K+-ATPase by NaCl with oligomycin or ATP

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+/K+-ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+-occluded E1 state of Na+/K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1 state, it is suggested that the Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between alpha subunits. Therefore, membrane-bound Na+/K+-ATPase would function as at least an (alphabeta)2-diprotomer with interacting alpha subunits at the phosphorylation step.