Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both 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/tau1 approximately 180 s-1 (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 rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity 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/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and 相似文献   

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.     

The effect of ionic strength and specific anions on substrate binding and hydrolytic activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3-, eosine2-, p-nitrophenylphosphate, and V(max) for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0-0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl approximately Na2SO4 approximately Na-acetate < NaNO3 < NaSCN < NaCl04. We treated the influence of NaCl and Na2SO4 on K(diss) for E x ADP as a "pure" ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3- effect was compatible with competitive binding of NO3- and ADP in addition to the general I-effect. K(diss) for E x NO3 was approximately 32 mM. Analysis of V(max)/K(m) for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.     

Photoinactivation of fluorescein isothiocyanate-modified Na,K-ATPase by 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate. Abolition of E1 and E2 partial reactions by sequential block of high and low affinity nucleotide sites

The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.     

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.     

Tissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase

The Na,K-ATPase comprises a catalytic alpha subunit and a glycosylated beta subunit. Another membrane polypeptide, gamma, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with alpha and beta in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-gamma antiserum to define the tissue specificity and topology of gamma and to address the question of whether gamma has a functional role. The trypsin sensitivity of the amino terminus of the gamma subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that gamma subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, alpha1-transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into gamma function, the effects of the anti-gamma serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that gamma stabilizes E1 conformation(s) of the enzyme and that anti-gamma counteracts this effect: (i) anti-gamma inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) --> E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-gamma indicating that the antibody shifts the E1 left and right arrow left and right arrow E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ concentration reduced to levels ( --> E2P transition, anti-gamma is stimulatory. These observations taken together with evidence that the pig gamma subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb+ ions partially protect it against this effect, indicate that the gamma subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-gamma disrupts alphabeta-gamma interactions and counteracts these modulatory effects of the gamma subunit.     

Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues

The functional roles of Asp804 and Asp808, located in the sixth transmembrane segment of the Na,K-ATPase alpha subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala808 --> Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEBS Lett. 389, 179-185). Asp804 and Asp808 were replaced by Ala, Asn, and Glu in the sheep alpha1 subunit and expressed in a mouse cell line where [3H]ouabain binding was utilized to probe the exogenous proteins. All of the heterologous proteins were targeted into the plasma membrane, bound ouabain and nucleotides, and adopted E1Na, E1ATP, and E2P conformations. K+ competition of ouabain binding to sheep alpha1 and Asp808 --> Glu enzymes displayed IC50 values of 4.11 mM (nHill = 1.4) and 23.8 mM (nHill = 1.6), respectively. All other substituted proteins lacked this K+-ouabain antagonism, e.g. 150 mM KCl did not inhibit ouabain binding. Na+ antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (nHill 相似文献   

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: cytoplasmic cation binding and release

In the preceding publication (. Biophys. J. 76:000-000) a new technique was described that was able to produce concentration jumps of arbitrary ion species at the surface of a solid supported membrane (SSM). This technique can be used to investigate the kinetics of ion translocating proteins adsorbed to the SSM. Charge translocation of the Na+/K+-ATPase in the presence of ATP was investigated. Here we describe experiments carried out with membrane fragments containing Na+/K+-ATPase from pig kidney and in the absence of ATP. Electrical currents are measured after rapid addition of Na+. We demonstrate that these currents can be explained only by a cation binding process on the cytoplasmic side, most probably to the cytoplasmic cation binding site of the Na+/K+-ATPase. An electrogenic reaction of the protein was observed only with Na+, but not with other monovalent cations (K+, Li+, Rb+, Cs+). Using Na+ activation of the enzyme after preincubation with K+ we also investigated the K+-dependent half-cycle of the Na+/K+-ATPase. A rate constant for K+ translocation in the absence of ATP of 0.2-0.3 s-1 was determined. In addition, these experiments show that K+ deocclusion, and cytoplasmic K+ release are electroneutral.     

Consequences of mutations to the phosphorylation site of the alpha-subunit of Na, K-ATPase for ATP binding and E1-E2 conformational equilibrium

Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.     

Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.     

Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600 s(-1) and 1.5 x 10(6) M(-1) s(-1), respectively, at pH 7.2 and 22 degrees C.     

Different susceptibility to phospholipase A2 treatment of the fluorescence intensity changes in the vicinity of Cys-964 and Lys-501 in the alpha-chain of probe-labeled Na+,K(+)-ATPase

Phospholipase A2 [EC 3.1.1.4] treatment of pig kidney Na+,K(+)-ATPase [EC 3.6.1.3] labeled with fluorescence probes at the alpha-chain reduced the extent of the fluorescence intensity change of an N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) probe at Cys-964 to below one-third of the control level accompanying the accumulation of phosphoenzymes. However, it only induced a slight decrease in that of a fluorescence isothiocyanate (FITC) probe at Lys-501 with a large decrease in the rate of change. The addition of phosphatidylserine (PS) or phosphatidylinositol (PI) to the phospholipase-treated BIPM-FITC-labeled enzyme increased the rate of the FITC fluorescence change. Phospholipase treatment of the BIPM-enzyme greatly reduced the Na+,K(+)-ATPase activity. The addition of PS or PI to the treated enzyme induced reactivation. These data and others suggest that Cys-964 and Glu-953 (Rb+ protectable dicyclohexyl carbodiimide binding site) are located in the vicinity of the surface area of the enzyme where hydrocarbon chains of phospholipids are present, and conserved H-bonding amino acids, Thr-955 and Ser-962, are located rather near the center of a domain forming a cation binding route or cage with other hydrophobic transmembrane segments. These data may indicate that the interaction between the BIPM probe and the hydrocarbon chains of phospholipids changes in such a way as to sense the change in the binding state of various ligands accompanying the sequential appearance of reaction intermediates of the enzyme.     

ATP1AL1, a member of the non-gastric H,K-ATPase family, functions as a sodium pump

The human ATP1AL1-encoded protein (an alpha subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase beta subunit (gH,Kbeta) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86Rb+-influx mediated by the ATP1AL1-gH,Kbeta heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same cells. In this study we find that the intracellular Na+ content in cells expressing ATP1AL1 and gH,Kbeta is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 microM ouabain. Moreover, analysis of net Na+ efflux in HEK 293 expressing the ATP1AL1-gH,Kbeta heterodimer reveals the presence of Na+ extrusion activity that is not sensitive to 1 microM ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na+ efflux in control HEK 293 cells is similarly sensitive to either 1 microM or 1 mM ouabain. Finally, 86Rb+ influx through the ATP1AL1-gH,Kbeta complex is comparable to the 1 mM ouabain-sensitive Na+ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase beta subunit in HEK 293 cells mediates primarily Na+,K+ rather than H+,K+ exchange.     

Changes in steady-state conformational equilibrium resulting from cytoplasmic mutations of the Na,K-ATPase alpha-subunit

Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

Na+,K(+)-ATPase of human placenta during gestational hypertension: a biochemical-biophysical study

1. Na+,K(+)-ATPase is the membrane enzyme catalysing the active transport of Na+ and K+ across the plasma membrane of animal cells. A reduced activity of Na+,K(+)-ATPase has been described in gestational hypertension in a variety of cell types, in agreement with the hypothesis that gestational hypertension can induce membrane transport modifications similar to those reported for essential hypertension. The causes of the reduced Na+,K(+)-ATPase activity are still debated. 2. The aim of the present work was to investigate the molecular mechanism of the reduced enzymic activity in gestational hypertension using as a model Na+,K(+)-ATPase purified from human placenta. Na+,K(+)-ATPase obtained from term placentas of eight healthy pregnant women and eight age-matched women with gestational hypertension was purified as previously described. 3. We observed in gestational hypertension: (i) a significant increase in the activation energies above transition temperature; (ii) a significant decrease in the fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (i.e. increased fluidity) and an increase in the mean lifetime (modified hydrophobicity); (iii) a lower Kq, suggesting an enzymic structural modification; and (iv) an increased mean lifetime and rotational relaxation time of pyrene isothiocyanate, indicating a modified ATP binding site.     

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.     

Toxicological effects of beryllium on platelets and vascular endothelium

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.