Chimeras of X+, K+-ATPases. The M1-M6 region of Na+, K+-ATPase is required for Na+-activated ATPase activity, whereas the M7-M10 region of H+, K+-ATPase is involved in K+ de-occlusion

In this study we reveal regions of Na(+),K(+)-ATPase and H(+),K(+)-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H(+),K(+)-ATPase was replaced by that of Na(+),K(+)-ATPase was phosphorylated in the absence of Na(+) and showed no K(+)-dependent reactions. Next, the part originating from Na(+),K(+)-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na(+),K(+)-ATPase, harbors the amino acids responsible for Na(+) specificity. Compared with Na(+),K(+)-ATPase, this chimera displayed a similar apparent Na(+) affinity, a lower apparent K(+) affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that the E(2)K form of this chimera is less stable than that of Na(+),K(+)-ATPase, suggesting that it, like H(+),K(+)-ATPase, de-occludes K(+) ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the beta-subunit are involved in K(+) occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na(+)-stimulated ATPase reaction of Na(+),K(+)-ATPase, while on the other hand it has the K(+) occlusion properties of H(+),K(+)-ATPase.     

Identification of potential regulatory sites of the Na+,K+-ATPase by kinetic analysis

Kinetic models are presented that allow the Na(+),K(+)-ATPase steady-state turnover number to be estimated at given intra- and extracellular concentrations of Na(+), K(+), and ATP. Based on experimental transient kinetic data, the models utilize either three or four steps of the Albers-Post scheme, that is, E(2) --> E(1), E(1) --> E(2)P (or E(1) --> E(1)P and E(1)P --> E(2)P), and E(2)P --> E(2), which are the major rate-determining steps of the enzyme cycle. On the time scale of these reactions, the faster binding steps of Na(+), K(+), and ATP to the enzyme are considered to be in equilibrium. Each model was tested by comparing calculations of the steady-state turnover from rate constants and equilibrium constants for the individual partial reactions with published experimental data of the steady-state activity at varying Na(+) and K(+) concentrations. To provide reasonable agreement between the calculations and the experimental data, it was found that Na(+)/K(+) competition for cytoplasmic binding sites was an essential feature required in the model. The activity was also very dependent on the degree of K(+)-induced stimulation of the reverse reaction E(1) --> E(2). Taking into account the physiological substrate concentrations, the models allow the most likely potential sites of short-term Na(+),K(+)-ATPase regulation to be identified. These were found to be (a) the cytoplasmic Na(+) and K(+) binding sites, via changes in Na(+) or K(+) concentration or their dissociation constants, (b) ATP phosphorylation (as a substrate), via a change in its rate constant, and (c) the position of the E(2)<==>E(1) equilibrium.     

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.     

Mutational analysis of the putative K(+)-binding site on the fourth transmembrane segment of the gastric H(+),K(+)-ATPase

By means of a functional expression system and site-directed mutagenesis, we analyzed the role of the putative K(+)-binding site, Glu-345, located in the fourth transmembrane segment of the gastric H(+),K(+)-ATPase alpha-subunit. In the present study, we used several mutants, with alanine, isoleucine, leucine, glutamine, valine, lysine, and aspartic acid instead of Glu-345, and analyzed the H(+),K(+)-ATPase partial reactions of the mutants to determine the precise role of this residue. All the mutants except E345Q exhibited no H(+),K(+)-ATPase activity. The E345Q mutant showed 3-times higher affinity for ATP. This mutation shifted the optimum pH toward a more alkaline one. The E345A, E345I, E345L, E345V as well as E345Q mutants were phosphorylated with ATP as in the case of the wild-type H(+),K(+)-ATPase, whereas the E345K mutant was not phosphorylated. The E345Q mutant was dephosphorylated in the presence of K(+), but its affinity for K(+) was significantly lower than that of the wild type. The E345A, E345I, E345L, and E345V mutants did not exhibit sensitivity to K(+) in the dephosphorylation step below 3 mM K(+). Therefore, Glu-345 is important for the conformational change induced by K(+), especially in the dephosphorylation step in which K(+) reacts with the enzyme from the luminal side with high affinity and accelerates the release of inorganic phosphate. The glutamic acid in the fourth transmembrane segment is conserved, and was found to be involved in the cation-induced conformational change in H(+),K(+)-ATPase as well as Na(+),K(+)-ATPase and Ca(2+)-ATPase, however, the precise roles of the side chain in the function were different.     

Importance of Glu(282) in transmembrane segment M3 of the Na(+),K(+)-ATPase for control of cation interaction and conformational changes

Glu(282) located in the NH(2)-terminal part of transmembrane helix M3 of the Na(+),K(+)-ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na(+),K(+)-ATPase: (i) the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme, (ii) Na(+) binding at the cytoplasmically facing sites of E(1), and (iii) long-range interaction controlling dephosphorylation. In mutants Glu(282) --> Lys and Glu(282) --> Asp, the E(1) form was favored during ATP hydrolysis, whereas the E(2) form was favored in Glu(282) --> Ala and Glu(282) --> Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na(+), at least 3-fold for Glu(282) --> Lys and Glu(282) --> Asp, suggesting a direct effect on the Na(+) binding properties of E(1). Glu(282) --> Ala and Glu(282) --> Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na(+) without K(+) ("Na(+)-ATPase activity"), because of an increased rate of dephosphorylation of E(2)P. These results are in accordance with the hypothesis that Glu(282) is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na(+).     

How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway

We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.     

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.     

Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity

Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.     

Involvement of sulfhydryl groups in the inhibition of brain (Na+ + K+)-ATPase by pyrithiamin

Brain (Na+ + K+)-ATPase was protected by low concentrations of GSH from the inhibitory effect of pyrithiamin. The possible involvement of sulfhydryl groups in the inhibition was then studied by comparing the effect of pyrithiamin with that of N-ethylmaleimide on the enzyme. The treatment of rat brain (Na+ + K+)-ATPase with thesee inhibitors caused a significant decrease in reactivity of the enzyme to N-ethyl[3H]maleimide. N-Ethylmaleimide, like pyrithiamin, inhibited the partial reactions of (Na+ + K+)-ATPase system in parallel with the inhibition of the overall reaction. An SDS-polyacrylamide gel electrophoresis procedure indicated that pyrithiamin and N-ethylmaleimide inhibited Na+-dependent phosphorylation of the alpha(+) form of rat brain (Na+ + K+)-ATPase more than that of alpha, though the selectivity for the alpha(+) seemed to be higher with the former inhibitor than in the latter. The treatment also decreased sensitivity of the enzyme to ouabain inhibition. However, pyrithiamin- and N-ethylmaleimide-induced inactivations of the enzyme differed in the efficacy of GSH for protection and in the effect of the kind of ligands present during the reaction. Furthermore, pyrithiamin did not appear to interact directly with sulfhydryl groups, but caused the formation of disulfide in bovine brain (Na+ + K+)-ATPase. In contrast to N-ethylmaleimide, pyrithiamin did not affect the sulfhydryl-enzymes such as alcohol dehydrogenase and L-alanine dehydrogenase. It is concluded that pyrithiamin modifies the functional sulfhydryl groups of brain (Na+ + K+)-ATPase in a way different from N-ethylmaleimide and causes a structural change and inactivation of the enzyme.     

Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

The Occlusion of Rb(+) in the Na(+)/K(+)-ATPase. II. The effects of Rb(+), Na(+), Mg2(+), or ATP on the equilibrium between free and occluded Rb(+).

We used the direct route of occlusion to study the equilibrium between free and occluded Rb(+) in the Na(+)/K(+)-ATPase, in media with different concentrations of ATP, Mg(2+), or Na(+). An empirical equation, with the restrictions imposed by the stoichiometry of ligand binding was fitted to the data. This allowed us to identify which states of the enzyme were present in each condition and to work out the schemes and equations that describe the equilibria between the ATPase, Rb(+), and ATP, Mg(2+), or Na(+). These equations were fitted to the corresponding experimental data to find out the values of the equilibrium constants of the reactions connecting the different enzyme states. The three ligands decreased the apparent affinity for Rb(+) occlusion without affecting the occlusion capacity. With [ATP] tending to infinity, enzyme species with one or two occluded Rb(+) seem to be present and full occlusion seems to occur in enzymes saturated with the nucleotide. In contrast, when either [Mg(2+)] or [Na(+)] tended to infinity no occlusion was detectable. Both Mg(2+) and Na(+) are displaced by Rb(+) through a process that seems to need the binding and occlusion of two Rb(+), which suggests that in these conditions Rb(+) occlusion regains the stoichiometry of the physiological operation of the Na(+) pump.     

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Nitrophenylphosphate as a tool to characterize gill Na, K-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.     

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).     

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.