The regulation of Na, K-ATPase activity by the substrate

The mechanism of functioning of Na, K-ATPase system is considered, the peculiarities of hydrolysis in different substrates are described. The experimental results testify to the role of substrate structure in E2----E1-transition, Na+ transport, K(+)-dependent phosphatase activity and quaternary structure of enzyme. The regulatory role of molecular organization of Na, K-ATPase in ion transport is discussed.     

N-acetylimidazole inactivates renal Na,K-ATPase by disrupting ATP binding to the catalytic site

Treatment of renal Na,K-ATPase with N-acetylimidazole (NAI) results in loss of Na,K-ATPase activity. The inactivation kinetics can be described by a model in which two classes of sites are acetylated by NAI. The class I sites are rapidly reacting, the acetylation is prevented by the presence of ATP (K0.5 congruent to 8 microM), and the inactivation is reversed by incubation with hydroxylamine. These data suggest that the class I sites are tyrosine residues at the ATP binding site. The second class of sites are more slowly reacting, not protected by ATP, nor reversed by hydroxylamine treatment. These are probably lysine residues elsewhere in the protein. The associated K-stimulated p-nitrophenylphosphatase activity is inactivated by acetylation of the class II sites only; thus the tyrosine residues associated with ATP binding to the catalytic center are not essential for phosphatase activity. Inactivated enzyme no longer has high-affinity ATP binding associated with the catalytic site, although low-affinity ATP effects (inhibition of phosphatase and deocclusion of Rb) are still present. The inactivated enzyme can still be phosphorylated by Pi, occlude Rb+ ions, and undergo the major conformational transitions between the E1 Na and E2 K forms of the enzyme. Thus acetylation of the Na,K-ATPase by NAI inhibits high-affinity ATP binding to the catalytic center and produces inactivation.     

The role of magnesium ions in the regulation of the catalytic activity of kidney Na+,K+-ATPase

Free Mg2+ is studied for its effect on the activation kinetics of pig kidney Na+, K+-ATPase by monovalent cations (nH and K0.5 for Na+ and K+ are determined). It is established that at the saturating concentration of complementary ion-activator an increase of free Mg2+ concentration up to 12 mM is accompanied by a rise of nH and K0.5 for Na+ and a fall of K0.5 for K+ without nH changes for this cation. The analysis of inhibition kinetics shows that free Mg2+ is a competitive inhibitor as to Na+ and noncompetitive as to K+. It is concluded that inhibition of Na+, K+-ATPase by free Mg2+ is a complex process including competition with Na+ at its binding sites and the "occluding" of enzyme at the stage, preceding dissociation of cation and also the weakening of subunit interactions in the enzyme.     

Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase

The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O-methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p-nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high-affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the (-K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the (-K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high-affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1).     

The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.     

Inactivation of Na,K-ATPase following Co(NH3)4ATP binding at a low affinity site in the protomeric enzyme unit

The Na(+)-dependent or E1 stages of the Na,K-ATPase reaction require a few micromolar ATP, but submillimolar concentrations are needed to accelerate the K(+)-dependent or E2 half of the cycle. Here we use Co(NH(3))(4)ATP as a tool to study ATP sites in Na,K-ATPase. The analogue inactivates the K(+) phosphatase activity (an E2 partial reaction) and the Na,K-ATPase activity in parallel, whereas ATP-[(3)H]ADP exchange (an E1 reaction) is affected less or not at all. Although the inactivation occurs as a consequence of low affinity Co(NH(3))(4)ATP binding (K(D) approximately 0.4-0.6 mm), we can also measure high affinity equilibrium binding of Co(NH(3))(4)[(3)H]ATP (K(D) = 0.1 micro m) to the native enzyme. Crucially, we find that covalent enzyme modification with fluorescein isothiocyanate (which blocks E1 reactions) causes little or no effect on the affinity of the binding step preceding Co(NH(3))(4)ATP inactivation and only a 20% decrease in maximal inactivation rate. This suggests that fluorescein isothiocyanate and Co(NH(3))(4)ATP bind within different enzyme pockets. The Co(NH(3))(4)ATP enzyme was solubilized with C(12)E(8) to a homogeneous population of alphabeta protomers, as verified by analytical ultracentrifugation; the solubilization did not increase the Na,K-ATPase activity of the Co(NH(3))(4)ATP enzyme with respect to parallel controls. This was contrary to the expectation for a hypothetical (alphabeta)(2) membrane dimer with a single ATP site per protomer, with or without fast dimer/protomer equilibrium in detergent solution. Besides, the solubilized alphabeta protomer could be directly inactivated by Co(NH(3))(4)ATP, to less than 10% of the control Na,K-ATPase activity. This suggests that the inactivation must follow Co(NH(3))(4)ATP binding at a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites.     

Role of protein conformation changes and transphosphorylations in the function of Na+/K(+)-transporting adenosine triphosphatase: an attempt at an integration into the Na+/K+ pump mechanism.

The particular aim of the review on some basic facets of the mechanism of Na+/K(+)-transporting ATPase (Na/K-ATPase) has been to integrate the experimental findings concerning the Na(+)- and K(+)-elicited protein conformation changes and transphosphorylations into the perspective of an allosterically regulated, phosphoryl energy transferring enzyme. This has led the authors to the following summarizing evaluations. 1. The currently dominating hypothesis on a link between protein conformation changes ('E1 in equilibrium with E2') and Na+/K+ transport (the 'Albers-Post scheme') has been constructed from a variety of partial reactions and elementary steps, which, however, do not all unequivocally support the hypothesis. 2. The Na(+)- and K(+)-elicited protein conformation changes are inducible by a variety of other ligands and modulatory factors and therefore cannot be accepted as evidence for their direct participation in effecting cation translocation. 3. There is no evidence that the 'E1 in equilibrium with E2' protein conformation changes are moving Na+ and K+ across the plasma membrane. 4. The allosterically caused ER in equilibrium with ET ('E1 in equilibrium with E2') conformer transitions and the associated cation 'occlusion' in equilibrium with 'de-occlusion' processes regulate the actual catalytic power of an enzyme ensemble. 5. A host of experimental variables determines the proportion of functionally competent ER enzyme conformers and incompetent ET conformers so that any enzyme population, even at the start of a reaction, consists of an unknown mixture of these conformers. These circumstances account for the occurrence of contradictory observations and apparent failures in their comparability. 6. The modelling of the mechanism of the Na/K-ATPase and Na+/K+ pump from the results of reductionistically designed experiments requires the careful consideration of the physiological boundary conditions. 7. Na+ and K+ ligandation of Na/K-ATPase controls the geometry and chemical reactivity of the catalytic centre in the cycle of E1 in equilibrium with E2 state conversions. This is possibly effected by hinge-bending, concerted motions of three adjacent, intracellularly exposed peptide sequences, which shape open and closed forms of the catalytic centre in lock-and-key responses. 8. The Na(+)-dependent enzyme phosphorylation with ATP and the K(+)-dependent hydrolysis of the phosphoenzyme formed are integral steps in the transport mechanism of Na/K-ATPase, but the translocations of Na+ and K+ do not occur via a phosphate-cation symport mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Occlusion of Na+ by the Na,K-ATPase in the presence of oligomycin

Oligomycin occludes Na+ in an E1-form of the Na,K-ATPase. The rate constants for the release of Na+ from the E1-form and for the transition to the E2-form are about 0.5 s-1. The effect of oligomycin is not seen using other cations which also have a Na+-like effect on the enzyme conformation. The inhibitory effect of oligomycin on the ADP-ATP dependent Na:Na exchange but not on the accompanying ADP-ATP exchange can be explained from a decrease in the rate of release of Na+ from an E1 approximately phosphoform with Na+ occluded, E'1 approximately P (Na3), i.e. with Na+ in the membrane phase, to an E"1 approximately PNa3 form with Na+ not occluded. E"1 approximately PNa3 is at a step before formation of E2-P, and disappears at a high rate when ADP reacts with E"1 approximately P (Na3).     

The conformation of H,K-ATPase determines the nucleoside triphosphate (NTP) selectivity for active proton transport

The gastric H,K-ATPase is related to other cation transport ATPases, for example, Na,K-ATPase and Ca-ATPase, which are called E1-E2 ATPases in recognition of conformational transitions during their respective transport and catalytic cycles. Generally, these ATPases cannot utilize NTPs other than ATP for net ion transport activity. For example, under standard assay conditions, rates of NTP hydrolysis and H+ pumping by the H,K-ATPase for CTP are about 10% of those for ATP and undetectable with GTP, ITP, and UTP. However, we observed that H,K-ATPase will catalyze NTP/ADP phosphate exchange at similar rates for all of these NTPs, suggesting that a common phosphoenzyme intermediate is formed. The present study was undertaken to evaluate the specificity of nucleotides to power the H,K-ATPase and several of its partial reactions, including NTP/ADP exchange, K+-catalyzed phosphatase activity, and proton pumping. Results demonstrate that under conditions that promote the conformational change of the K+ bound form of the enzyme, K.E2, to E1, all NTPs tested support K+-stimulated NTPase activity and H+ pumping up to 30-50% of that with ATP. These conditions include (1) the presence of ADP as well as the NTP energy source and (2) reduced K+ concentration on the cytoplasmic side to approximately 0. These data conform to structural models for E1-E2 ATPases whereby adenosine binding promotes the K.E2 to E1 conformational change and K+ deocclusion.     

A model for the reaction pathways of the K+-dependent phosphatase activity of the (Na+ + K+)-dependent ATPase

(Na+ + K+)-dependent ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+ -dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: (formula; see text) Differences between the three preparations in K0.5 for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in K0.5 produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+ -dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+ -sites are oriented extracellularly, as are those of the (Na+ + K+)-dependent ATPase reaction. Dimethyl sulfoxide accentuated Na+ -stimulated K+ -dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+ -sites of the Na+ -stimulated pathway.     

Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.     

Evidence for a relationship between activity and the tetraprotomeric assembly of solubilized pig gastric H/K-ATPase

Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C12E8) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5'-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated by a single-molecule detection technique using total internal reflection fluorescence microscopy. Solubilization of the FITC-H/K-ATPase reduced the potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity to around 5% of the level of the membrane-bound enzyme with the formation of 50% protomer and 40% diprotomer. The solubilization of unmodified H/K-ATPase also reduced both the K-pNPPase and H/K-ATPase activities to around 5%. However, solubilization with increasing concentrations of potassium acetate induced significant and similar increases in K-pNPPase activity (K0.5 = 35 mM) with an increase in the amount of the tetraprotomer of FITC-H/K-ATPase, and the K-pNPPase (K0.5 = 28 mM) and H/K-ATPase (K0.5 = 40 mM) activities of the unmodified H/K-ATPase. The correlation coefficient between the proportion of tetraprotomer and the proportion of the K-pNPPase activity for the same FITC-H/K-ATPase preparation was estimated to be 0.93. Similar coefficients were also obtained between the proportion of tetraprotomer in the FITC-H/K-ATPase and the proportion of K-pNPPase and H/K-ATPase activities in the unmodified H/K-ATPase, with value of 0.85 and 0.86, respectively. Such positive correlations were not obtained between these activities and other oligomeric species. These data, the first direct comparison of oligomeric assembly and enzyme activity both stabilized by K+ in C12E8-solubilized gastric H/K-ATPase, provide strong evidence that the catalytic unit of C12E8-solubilized gastric H/K-ATPase is a tetraprotomer.     

Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain- sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.     

Uncoupling the red cell sodium pump by proteolysis

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.     

Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.     

Binding of Na+ ions to the Na,K-ATPase increases the reactivity of an essential residue in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI), a new imidazole-based probe, results in irreversible loss of enzymatic activity. Inactivation of 95% of the Na,K-ATPase activity is achieved by the covalent binding of 1 molecule of [3H]NIPI to a single site on the alpha-subunit of the Na,K-ATPase. The reactivity of this site toward NIPI is about 10-fold greater when the enzyme is in the E1Na or sodium-bound form than when it is in the E2K or potassium-bound form. K+ ions prevent the enhanced reactivity associated with Na+ binding. Labeling and inactivation of the enzyme is prevented by the simultaneous presence of ATP or ADP (but not by AMP). The apparent affinity with which ATP prevents the inactivation by NIPI at pH 8.5 is increased from 30 to 3 microM by the presence of Na+ ions. This suggests that the affinity with which native enzyme binds ATP (or ADP) at this pH is enhanced by Na+ binding to the enzyme. Modification of the single sodium-responsive residue on the alpha-subunit of the Na,K-ATPase results in loss of high affinity ATP binding, without affecting phosphorylation from Pi. Modification with NIPI probably alters the adenosine binding region without affecting the region close to the phosphorylated carboxyl residue aspartate 369. Tightly bound (or occluded) Rb+ ions are not displaced by ATP (4 mM) in the inactivated enzyme. Thus modification of a single residue simultaneously blocks ATP acting with either high or low affinity on the Na,K-ATPase. These observations suggest that there is a single residue on the alpha-subunit (probably a lysine) which drastically alters its reactivity as Na+ binds to the enzyme. This lysine residue is essential for catalytic activity and is prevented from reacting with NIPI when ATP binds to the enzyme. Thus, the essential lysine residue involved may be part of the ATP binding domain of the Na,K-ATPase.     

Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ +K+)-dependent ATPase

Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.     

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither in the presence nor in the absence of anionic phospholipids did protein kinase phosphorylation of native FXYD10, which relieves the inhibition, affect such cross-linking. Together, this seems to indicate that phosphorylation involves only modest structural rearrangements between the cytoplasmic domain of FXYD10 and the Na,K-ATPase A-domain.     

Regulation of the frontocortical sodium pump by Na+ in Alzheimer's disease: difference from the age-matched control but similarity to the rat model

The Na+ and K+ dependence of the frontocortical Na,K-ATPase in Alzheimer's disease (AD) was compared with that in human control (Co) and rat AD model. In AD, the relationship between the Na/K ratio and the Na,K-ATPase activity showed noticeable left-shift with three-fold increase in the enzyme affinity for Na+ (K(0.5)=10 and 30 mM in AD and Co, respectively). The Na+ dependence of the enzyme in AD showed two different Hill coefficients (n(H)), 1.1 and 0.3, whereas the Co value of n(H) was higher (1.4). The rat AD model generated by ibotenic acid revealed a Na+ dependence similar to AD. The K+ dependence of the Na,K-ATPase showed no significant difference in AD and Co. Compared with Co, AD produced a shift in the break of the Na,K-ATPase Arrhenius plot, suggesting remarkable alterations in the enzyme lipid environment. Our findings support the hypothesis that dysfunction of the Na,K-ATPase in AD is provoked by altered Na+ dependence of the enzyme. An impairment of the pump functionality might serve as an early mechanism of AD that should be interrupted by selective pharmacological agents.