Conformational change accompanying formation of oligomycin-induced Na(+)-bound forms and their conversion to ADP-sensitive phosphoenzymes in Na+,K(+)-ATPase.

Oligomycin reduced the fluorescence intensity of an N-(p-(2-benzimidazoly)phenyl) maleimide (BIPM) probe at Cys-964 of the alpha-chain of pig kidney Na+,K(+)-ATPase with increase in the concentration of Na+ with a Hill coefficient of nh = 0.77 with Kh = 231 mM. The maximum fluorescence decrease was around 80% of the value observed after accumulation of ADP-sensitive phosphoenzyme (E1P) in the presence of 2 M Na+. The addition of Mg2+ and ATP with Na+ or choline chloride to give the same final ligand concentration to the Na(+)-enzyme-oligomycin complex formed with 16 mM Na+ + 1,984 mM choline chloride or 2 M Na+ induced rapid phosphorylation (20 or 21/s) and slower fluorescence decrease (12.1 +/- 1.2 or 10.1 +/- 3.2/s). These additions to the Na(+)-enzyme complex formed under the former or the latter conditions induced slow phosphorylation (13/s) prior to a much slower fluorescence decrease (3.4 +/- 0.3 or 8.6 +/- 0.7/s). The addition of Ca2+ and ATP to these enzyme complexes induced rapid fluorescence changes (21-11/s) followed by one order of magnitude slower rates of phosphorylation (1.5-1.3 s). These data suggest that the decrease in BIPM fluorescence induced by ATP with Ca2+ or with Mg2+, reflects the change of the Na+ binding state before or after the formation of E1P, respectively.     

Conformational change of sodium- and potassium-dependent adenosine triphosphatase. Conformational evidence for the Post-Albers mechanism in Na+- and K+-dependent hydrolysis of ATP

The addition of ATP with K+ to pig kidney Na+,K+-ATPase (EC 3.6.1.3) modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide induced a transient decrease (t 1/2 = 0.01 s) in the fluorescence in the presence of Mg2+ with 0.64 M Na+, followed by a slow increase (t 1/2 = 0.08 s), to give a higher steady level than that observed without K+. The addition induced a transient increase (t 1/2 less than 0.02 s) in the amount of phosphoenzyme, followed by a slow decrease (t 1/2 = 0.08 s), but the addition without K+ induced a monophasic increase (t 1/2 = 0.02 s). The addition of ATP in the presence of 2 M Na+ with Ca2+ induced a monophasic decrease (t 1/2 = 0.1 s) in the fluorescence along with a much slower increase (t 1/2 = 1.2 s) in the amount of phosphoenzyme. No significant burst of acid-labile phosphate was observed. The data showed clearly the accumulation of the enzyme-ATP complex preceding the phosphoenzyme formation. Fluorescence intensity of these enzyme species and the amount of phosphoenzyme permitted the simulation using the reaction mechanism including enzyme-ATP complex, ADP-sensitive phosphoenzyme, K+-sensitive phosphoenzyme, and K+-bound enzyme. The simulation gave a good fit to the experimental data which showed that ATP is hydrolyzed in sequence through the above intermediates in the presence of both Na+ and K+.     

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

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

Electrical potential accelerates the E1P(Na)----E2P conformational transition of (Na,K)-ATPase in reconstituted vesicles

We have used renal (Na,K)-ATPase, covalently labeled with fluorescein, and phospholipid vesicles reconstituted with labeled enzyme, to detect conformational transitions induced by acetyl phosphate in the presence of Mg2+ and Na+ ions. Equilibrium fluorescence measurements show quenching of the fluorescein fluorescence, which is thought to reflect conversion of the initial E1 form to the phosphorylated E2P form. These fluorescence changes occur on inside-out-oriented pumps. The rates of acetyl phosphate-induced fluorescence changes have been measured using a stopped-flow fluorimeter. The rate of fluorescence quenching (1.5-3 s-1) is a measure of the rate of the E1P(Na)----E2P transition. The quenching is preceded by a fast fluorescence increase (12.3 +/- 4 s-1) associated with phosphorylation of E1 to E1P(Na), shown clearly in experiments with enzyme treated with oligomycin. Oligomycin greatly reduces the rate of the fluorescence quenching (0.044 +/- 0.01 s-1). Using potassium-loaded vesicles treated with valinomycin or lithium-loaded vesicles treated with Li+ ionophore N,N'-diheptyl-N,N'-didiethyl ether, 5,5-dimethyl-3,7-dioxanonanediamide in order to induce electrical diffusion potentials, negative inside, the rates of the fluorescence quenching are accelerated by up to 4-fold. The experiments demonstrate that the conformational transition E1P(Na)----E2P, associated with transport of 3 Na+ ions, is a voltage-sensitive reaction, carrying a net positive charge. This confirms a prediction based on transport experiments. In experiments with fluorescein-labeled (Na,K)-ATPase, the use of acetyl phosphate rather than ATP, which does not bind, provides a valuable tool to detect fluorescence signals accompanying steps in the turnover cycle.     

Acceleration of the rate of fluorescence decrease by high concentrations of ATP under the condition of accumulation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase

Addition of up to 300 microM ATP in the presence of 2 M NaCl with MgCl2 to pig kidney Na+,K+-ATPase treated with N-[p-(2-benzimidazolyl)phenyl]maleimide seemed to be insufficient to saturate the rate of the fluorescence decrease. However, both the extent of the decrease and the amount of phosphoenzyme at a steady state were saturated below 20 microM ATP. Addition of Mg2+ with Na+ to the enzyme preincubated with 20 to 600 microM ATP gave nearly the same rate constant, which was below 50% of that obtained by adding 300 microM ATP to the Na+-form enzyme in the presence of Mg2+. High concentrations of ATP affected neither the rate of light-scattering change (Taniguchi, K. et al. (1986) J. Biol. Chem. 261, 3272-3281) after ADP-sensitive phosphoenzyme formation (E1P) nor that of the breakdown of E1P. A stoichiometric amount of [32P]Pi was liberated from [32P]E1P. The data suggested that ATP did not bind to E1P in such a way as to increase the extent of phosphorylation further or to accelerate dephosphorylation. The data also suggested that the reason for the large difference in the apparent affinity of ATP as evaluated from the rate and the extent of fluorescence change is the large dissociation constant for ATP of a Michaelis complex.     

5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover

The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively.     

Breakdown of Na+/K+-exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+-ATPase activity.

The reactivity towards Na+ and K+ of Na+/K+-ATPase phosphoenzymes formed from ATP and Pi during Na+-ATPase turnover and that obtained from Pi in the absence of ATP, Na+ and K+ was studied. The phosphoenzyme formed from Pi in the absence of cycling and with no Na+ or K+ in the medium showed a biphasic time-dependent breakdown. The fast component, 96% of the total EP, had a decay rate of about 4 s(-1) in K+-free 130 mm Na+, and was 40% inhibited by 20 mm K+. The slow component, about 0.14 s(-1), was K+ insensitive. Values for the time-dependent breakdown of the phosphoenzymes obtained from ATP and from Pi during Na+-ATPase activity were indistinguishable from each other. In K+-free medium containing 130 mm Na+, the decays followed a single exponential with a rate constant of 0.45 s(-1). The addition of 20 mm K+ markedly increased the decays and made them biphasic. The fast components had a rate of approximately 220 s-1 and accounted for 92-93% of the total phosphoenzyme. The slow components decayed at a rate of about 47-53 s(-1). A second group of experiments examined the reactivity towards Na+ of the E2P forms obtained with ATP and Pi when the enzyme was cycling. In both cases, the rate of dephosphorylation was a biphasic function of [Na+]: inhibition at low [Na+], with a minimum at about 5 mm Na+, followed by recovery at higher [Na+]. Although qualitatively similar, the phosphoenzyme formed from Pi showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na+-ATPase turnover share the same intermediates.     

Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells

Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chem., 243:1957), the phosphoenzyme formed at 0 degrees C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested that the conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1 approximately P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0 degrees C. Since the ATP:ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1 approximately P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0 degrees C. Simple saturation was observed instead with a K0.5 for Na of approximately 1 mM. The effect of increasing temperature on the ATP:ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0 degrees C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0 degrees C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 degrees C, there was an approximately 300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the E1 approximately P----E2P transition of the Na pump in human red cell membranes is blocked at 0 degrees C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP:ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0 degrees C, where E1 approximately P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme.     

ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase

In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by N?rby et al. (N?rby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.     

Evidence for essential carboxyls in the cation-binding domain of the Na,K-ATPase.

Treatment of isolated canine renal Na,K-ATPase with a stable diazomethane analog, 4-(diazomethyl)-7-(diethylamino)-coumarin (DEAC), results in enzyme inactivation. The inactivation rate was dramatically increased when the enzyme was treated with DEAC in the presence of ATP and Mg2+ (in imidazole buffer) or Pi and Mg2+, conditions which produce enzyme phosphorylation. Inactivation in the presence of Pi and Mg2+ could be partially prevented by Na+ and almost completely prevented by K+. The quantity of DEAC covalently bound to the Na,K-ATPase was determined spectrophotometrically. The extent of inactivation was linearly related to the amount of K-protectable DEAC incorporation. Complete inactivation of ATPase activity occurred with 2.14 +/- 0.18 nmol of DEAC covalently bound/mg of protein. This suggests that only 1 or 2 carboxyl residues/catalytic center (estimated by high affinity ADP binding) are involved in the modification leading to inactivation. The modified enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding, thus, the nucleotide-binding domain of the enzyme seems unaffected by the modification. In contrast, under conditions where native enzyme was able to occlude 3.82 nmol of K+ ions/mg of protein, DEAC-modified enzyme occluded only 0.33 nmol of K+ ions. Na+ occlusion by the enzyme (in the presence of oligomycin) was also reduced (by 80%) following treatment with DEAC. Phosphorylation by [32P]inorganic phosphate and Na(+)-activated phosphorylation of the modified enzyme with [32P]ATP yielded reduced levels of phosphoenzyme (about 36%) compared to native enzyme. The DEAC-modified [32P]phosphoenzyme formed from [32P]ATP was insensitive to the addition of K+ ions, under conditions which led to the rapid hydrolysis of native phosphoenzyme. Gel electrophoresis of modified protein revealed strong fluorescence labeling of the alpha-subunit, which was substantially reduced if treatment with DEAC was performed in the presence of K+ ions. Partial tryptic digestion and electrophoretic analysis revealed normal degradation patterns in the presence of ADP (E1 form) but the typical patterns, seen with K+ ions (E2K) or Na+ ions (E1Na) in native enzyme, were absent. A typical E2-like tryptic degradation pattern was seen, however, in the presence of vanadate ions and ouabain, suggesting that the modification does not freeze the enzyme in an E1 conformation and that the enzyme is still able to undergo the E1E2 conformational transition after modification. Our results suggest that a small number of carboxyl residues in the sodium pump alpha-subunit (perhaps one) are essential for K+ and Na+ binding and stabilizing the occluded enzyme cation forms. Esterification of the carboxyl groups by DEAC inactivates the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

The reaction sequence of the Na+/K(+)-ATPase: rapid kinetic measurements distinguish between alternative schemes.

Conformational changes between E1 and E2 enzyme forms of a dog kidney Na+/K(+)-ATPase preparation labeled with 5-iodoacetamidofluorescein were followed with a stopped-flow fluorimeter, in terms of the rate constant, kobs, and the steady-state magnitude, % delta F of fluorescence change. On rapid mixing of enzyme plus Mg2+ plus Na+ with saturating (0.5 mM) ATP in the absence of K+, kobs varied with Na+ concentration in the range 0-155 mM, with a K1/2 of 10 mM, while % delta F was relatively insensitive to Na+, with a K1/2 of 0.5 mM. Oligomycin reduced kobs by 98-99% for Na+ greater than or equal to 10 mM, but only by 50% for Na+ = 1 mM; % delta F was reduced at most by 20%. At 155 mM Na+, both kobs and % delta F changed if K+ was present with the enzyme. kobs decreased by 50% when K+ was increased from 0 to 0.2 mM, but increased when K+ was varied in the range 0.2-5 mM. K+ increased % delta F by a factor of 3 with a K1/2 of 0.3-0.5 mM as measured in both stopped-flow and steady-state experiments. These data are considered in terms of the derived presteady-state equations for two alternate schemes for the enzyme, with the E1P to E2P conformational change either preceding (Albers-Post) or following (N?rby-Yoda-Skou) Na+ transport and release. The analysis indicates that: (i) Na+ must be released before the conformational transition, from an E1 form; (ii) the step in which the second and/or third Na+ is released is rate-limiting, but this release is accelerated by Na+; and (iii) the release is also accelerated by K+ acting with low affinity (possibly at extracellular sites).     

The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.     

Conformational changes of renal sodium plus potassium ion-transport adenosine triphosphatase labeled with fluorescein

We studied conformational changes of purified renal sodium plus potassium ion-transport adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) labeled with fluorescein isothiocyanate. Fluorescein covalently binds to the alpha-subunit of the enzyme and inhibits the ATPase but not the p-nitrophenylphosphatase activity. Four unphosphorylated and three phosphorylated conformations were distinguished by the level of fluorescence and by the rate of its change (relative fluorescence is shown in percentages). Fluorescence of the ligand-free form (E1, 100%) was increased by Na+ (E1.Na form, 103%) and quenched by K+ (E2.K, 78%) at a site of high affinity (K0.5 for K+ = 0.07 mM). Mg2+ did not alter fluorescence of E1 or E1.Na but raised that of E2.K (E2.K.Mg form, 85-90%). Addition of excess Na+ to the E2.K.Mg form restored high fluorescence but the rate of transition from E2.K.Mg to E1.Na became progressively slower with increasing Mg2+ concentration. Two phosphorylated conformations, (E2-P).Mg (82%) and (E2-P).Mg.K (82%) were differentiated by a faster turnover of the latter form. A third conformation, (E2-P).Mg.ouabain, had the lowest fluorescence (56%) and its formation allowed the binding of ouabain to the phosphoenzyme. Reversible blocking of sulfhydryl groups with thimerosal inhibited the formation of E2.K and (E2-P).Mg.ouabain but not that of the other conformations of the fluorescein-enzyme. The thimerosal-treated fluorescein-enzyme retained K+-p-nitrophenylphosphatase activity, inhibition of this activity by ouabain and ouabain binding. The unphosphorylated enzyme had low (K0.5 = 1.2 mM) and the phosphoenzyme had high affinity (K0.5 = 0.03 - 0.09 mM) for Mg2+ in the absence of nucleotides. Since low and high affinity for Mg2+ alternates as the enzyme turns over, Mg2+ may be bound and released sequentially during the catalytic cycle.     

Side-specific effects of sodium on (Na,K)-ATPase. Studies with inside-out red cell membrane vesicles.

Using inside-out vesicles of human red cell membranes, the side-specific effects of Na+ on phosphorylation of (Na,K)-ATPase have been studied using low concentrations of [gamma-32P]ATP (less than or equal to 0.1 microM). Phosphorylation is stimulated by Na+ at the cytoplasmic membrane surface (extravesicular Na+) alone and not by Na+ at the external surface (intravesicular Na+). At 37 degrees C, external Na+ (less than or equal to 10 mM) does, however, increase the steady state level (approximately 2 1/2-fold) of phosphoenzyme above that observed with cytoplasmic Na+ alone; hydrolysis is increased to only a small extent. Little stimulation by external Na+ is observed at 0 degrees C. As Na+ at the cytoplasmic side is decreased to very low levels (less than or equal to 0.2 mM) several kinetic changes are observed: (i) the apparent turnover of phosphoenzyme (ratio Na+-ATP-ase/phosphoenzyme level) is markedly increased (approximately 3-fold, (ii) Rbext sensitivity (inhibition of (Na)-ATPase at low ATP levels) is reduced, and (iii) the ratio of Na+ ions transported per molecule of ATP hydrolyzed is decreased. These results are compatible with a reaction pathway involving a transition from one form of phosphoenzyme, E1-P, to another, E2-P of which the hydrolysis is decreased by moderate levels of external Na+. It is suggested also that an alternate reaction pathway for Na+-ATPase occurs at very low cytoplasmic Na+, one via hydrolysis of E1-P and not associated with Na+ translocation.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Conformational change accompanying transition of ADP-sensitive phosphoenzyme to potassium-sensitive phosphoenzyme of (Na+,K+)-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide

The addition of Mg2+ or ATP to (Na+,K+)-ATPase (EC 3.6.1.3) of pig kidney modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide simply reduced fluorescence in the presence of Na+; however, the addition of both ligands to the enzyme induced a reversible dynamic change. The direction of the change was dependent on the concentration of Na+ present. These dynamic changes in fluorescence intensity both in the presence of low and high concentrations of Na+ can be repeated by the re-addition of ATP but not by ADP. Addition of ouabain under the former condition stabilized the fluorescence at the highest level, but the addition of ouabain under the latter condition increased the fluorescence from the lowest to the highest level. The phosphoenzyme formed under the former condition was sensitive to K+ and insensitive to ADP while the phosphoenzyme formed under the latter condition was sensitive to ADP and insensitive to K+. The data indicate that the positive and negative fluorescence changes were induced by the formation of K+-sensitive phosphoenzyme and ADP-sensitive phosphoenzyme, respectively. N-Ethylmaleimide treatment partially inhibited the positive change without affecting the negative change. These data also indicate that the transition of ADP-sensitive phosphoenzyme to K+-sensitive phosphoenzyme accompanied the largest fluorescence intensity change which was examined during the hydrolysis of ATP. The data obtained from the tryptophan fluorescence of both the native and the modified enzyme suggest that the micro-environments of the tryptophan and the sulfhydryl residues are similar in the state of K+-sensitive phosphoenzyme but different in the state of ADP-sensitive phosphoenzyme.     

Protons as substitutes for sodium and potassium in the sodium pump reaction

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.     

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

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