ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

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.     

Sodium ion discharge from pig kidney Na+, K+-ATPase Na+-dependency of the E1P-E2P equilibrium in the absence of KCl

The two phosphoenzymes (E1P and E2P) of Na+,K+-ATPase were measured as ADP-sensitive and K+-sensitive fractions. The sum of these fractions was nearly 1 in the range of 50 to 1,200 mM NaCl. The effects of Na+ on the levels of E1P and E2P, on the rate constant of E2P leads to E1P transition (k2), on the rate constant of E2P dephosphorylation (k3), on the rate constant of E1P leads to E2P transition (k1) and on the apparent equilibrium constant between E1P and E2P (Kapp) were examined. k1 was found to decrease with increasing Na+ concentration, whereas k2 increased. Kapp was found to be directly proportional to the third power of Na+ concentration. k3 increased with increasing Na+ concentration and saturated at about 1 M NaCl. These results are consistent with a simple model in which ATP hydrolysis occurs through effectively only two phosphoenzyme intermediates in the absence of K+ and three sodium ions are discharged cooperatively from the enzyme during the E1P leads to E2P conversion.     

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+.     

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+.     

Phosphorylation of Na+, K(+)-ATPase by ATP in the presence of K+ and dimethylsulfoxide but in the absence of Na+

Purified Na+, K(+)-ATPase was phosphorylated by [gamma-32P]ATP in a medium containing dimethylsulfoxide and 5 mM Mg2+ in the absence of Na+ and K+. Addition of K+ increased the phosphorylation levels from 0.4 nmol phosphoenzyme/mg of protein in the absence of K+ to 1.0 nmol phosphoenzyme/mg of protein in the presence of 0.5 mM K+. Higher velocities of enzyme phosphorylation were observed in the presence of 0.5 mM K+. Increasing K+ concentrations up to 100 mM lead to a progressive decrease in the phosphoenzyme (EP) levels. Control experiments, that were performed to determine the contribution to EP formation from the Pi inevitably present in the assays, showed that this contribution was of minor importance except at high (20-100 mM) KCl concentrations. The pattern of EP formation and its KCl dependence is thus characteristic for the phosphorylation of the enzyme by ATP. In the absence of Na+ and with 0.5 mM K+, optimal levels (1.0 nmol EP/mg of protein) were observed at 20-40% dimethylsulfoxide and pH 6.0 to 7.5. Addition of Na+ up to 5 mM has no effect on the phosphoenzyme level under these conditions. At 100 mM Na+ or higher the full capacity of enzyme phosphorylation (2.2 nmol EP/mg of protein) was reached. Phosphoenzyme formed from ATP in the absence of Na+ is an acylphosphate-type compound as shown by its hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the alpha-subunit of the Na+, K(+)-ATPase as demonstrated by acid polyacrylamide gel electrophoresis followed by autoradiography.     

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

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

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.     

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.     

Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme

The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates

(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM. (2) In the absence of ADP the dephosphorylation curve was linear in a semilogarithmic plot almost from t = 0, whereas by addition of ADP a biphasic behaviour was obtained. The slope of the slow phase of dephosphorylation was virtually independent of the ADP concentration. (3) The results were analysed by the mathematical equation corresponding to the simplest possible model for the interconversion and breakdown of the phosphointermediates: (formula: see text) where alpha, beta, H and G are functions of all the rate constants and H and G furthermore are functions of the initial values for [E1P] and [E2P]. (4) The analysis confirmed the model and enabled the determination of all the rate constants. (5) k-1 was found to be equal to k'-1 + k"-1 . [ADP] indicating an ADP-independent 'spontaneous' dephosphorylation of E1P. The rate constant for this process was close to that for dephosphorylation of E2P, i.e., k'-1 congruent to k3. Also the value of k"-1 was determined. (6) k3 was found to be at least 10 . k-2. The implication of this for the role of the E1P to E2P transition in the Na+ + K+)-stimulated ATP hydrolysis will be discussed in detail in the following paper (Plesner, I.W., Plesner, L., N?rby, J.G. and Klodos, I. (1981) Biochim. Biophys. Acta 643, 483--494). (7) A refinement of the model, accounting for the effect of Na+ on the steady-state ratio between [E1P] and [E2P] is proposed: (formula: see text). At [Na+] = 150 mM as used here, E1P(Na) and E'1P are assumed to be in rapid equilibrium. (8) Comparison of our results with those of others underlines the general validity of the conclusions of the present paper.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

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.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

Existence of ADP- and KCl-insensitive phosphoenzyme intermediate of Na+,K(+)-ATPase at alkaline Ph

The Na(+)-ATPase activity of Na+,K(+)-ATPase in the absence of K+ was least dependent on the sodium concentration when the pH was 9.5. Around 40% of the phosphoenzyme formed from ATP in the presence of 0.5 mM MgCl2 at alkaline pH was insensitive to both KCl and ADP. High-Na+ chase reversed this insensitivity, i.e., the phosphoenzyme became sensitive to KCl or ADP. On the other hand, phosphorylation at 0.1 mM MgCl2 instead of 0.5 mM showed at least 95% sensitivity to KCl. These observations suggest that ADP- and KCl-insensitive phosphoenzyme was formed when excess Mg++ was present during phosphorylation at alkaline pH. This phosphoenzyme might be an intermediate in the process of ATP hydrolysis.     

Mathematical modelling of ATP, K+ and Na+ interactions with (Na+ + K+)-ATPase occurring under equilibrium conditions.

The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.     

Modulation of Na+-K+-ATPase by calcium ions

The modulatory effects of calcium ions on highly active Na+, K(+)-ATPase from calf brain and pig kidney tissues have been studied. The inhibitory action of Ca2+free on this enzyme depends on the level of ATP (but not AcP). The reduction of pH from 7.4 to 6.0 noticeably increases, but the elevation of pH to 8.0, in its turn, decreases the inhibition of ATP-hydrolyzing activity by calcium. With the increase of K+ concentration (in contrast to Na+) the sensibilization of Na+, K(+)-ATPase to Ca ions is observed. In the presence of potassium ions Mg2+free effectively modifies the inhibitory action of Ca2+free on this enzyme. Ca2+free (0.16-0.4 mM) decreases the sensitivity of Na+, K(+)-ATPase to action of the specific inhibitor ouabain in the presence of ATP. In the presence of AcP (phosphatase reaction) such a change of enzyme sensitivity to ouabain isn't observed. The influence of membranous effects of Ca2+ on the interaction of Na+, K(+)-ATPase with the essential ligands and cardiosteroids is discussed.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Difference between neuronal and nonneuronal (Na+ + K+)-ATPases in their conformational equilibrium

Several experiments were carried out to study the difference between two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase in the conformational equilibrium. Rat brain (Na+ + K+)-ATPase was much more thermolabile than the kidney enzyme. Both enzymes were protected from heat inactivation not only by Na+ and K+, but also by choline in varying degrees, though there was a difference between the two enzymes in the protection by the ligands. The brain enzyme was partially protected from N-ethylmaleimide (NEM) inactivation by both Na+ and K+, but the effects of the ligands on NEM inactivation of the kidney enzyme were more complex. Though ligands differentially affected the thermostability and NEM sensitivity of the two enzymes, the effects were not simply related to the conformational states. The sensitivity of phosphoenzyme (EP) formed in the presence of ATP, Na+, and Mg2+ to ADP or K+ and K+-p-nitrophenyl phosphatase (pNPPase) was then studied as a probe of the differences in the conformational equilibrium between the two isozymes. The EP of the brain enzyme was partially sensitive to ADP, while those of the heart and kidney enzymes were not. At physiological Na+ concentrations the percentages of E1P formed by the brain and kidney enzymes were determined to be about 40-50 and 10-20% of the total EP, respectively. The hydrolytic activity of pNPP in the presence of Li+, a selective activator at catalytic sites of the reaction, was much higher in the kidney enzyme than in the brain enzyme. The inhibition of K+-stimulated pNPPase by ATP and Na+ was greater in the latter enzyme than in the former. These results suggest that neuronal and nonneuronal (Na+ + K+)-ATPases differ in their conformational equilibrium: the E1 or E1P may be more stable in the alpha(+) than in the alpha during the turnover, and conversely the E2 or E2P may be more stable in the latter than in the former.     

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.