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

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

An attachment for nondestructive, fast quenching of samples in rapid-mixing experiments.

The present paper describes a quenching-and-washing chamber (QWC) to be used with a rapid-mixing apparatus (RMA) for the study of processes in the millisecond time scale. The QWC enables fast, nondestructive quenching by cooling and dilution of reactants in particulate systems that can be trapped on a filter. The reaction mixture (e.g., at 25 degrees C) is injected from the RMA into the QWC where it is immediately mixed with a stream of ice-cold solution flowing at a rate of 15-40 ml s-1. Quenching requires that the process studied is slowed considerably by cooling to 0-2 degrees C and/or by removal of reactants by dilution. The equipment was characterized through a study of the tight binding (occlusion) of 86Rb+ to purified, membrane-bound Na+/K+-ATPase. Millipore filters of 0.22-0.80 microm pore size trapped close to 100% of the enzyme protein. Enzyme with occluded 86Rb+ was formed in the RMA under conditions where the rate constant for release of Rb+ at 25 degrees C is up to 25 s-1 and then injected into the QWC. The high off-rate constant is due to the presence of 2.5 mM ATP, which accelerates release of Rb+. The recovery of occluded 86Rb+ on the filter was at least 90%, indicating that both cooling of the reactants and dilution of ATP are fast enough to stop the reaction. The quenching time was 3-4 ms.

An unexpected effect of ATP on the ratio between activity and phosphoenzyme level of Na+/K(+)-ATPase in steady state.

According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K(+)-ATPase requires the sequential formation of at least two conformers of a phosphoenzyme (E1P and E2P), followed by the K(+)-stimulated hydrolysis of E2P. In this paper we show that this model is a particular case of a more general class of models in all of which the ratio between ATPase activity (v) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers and of dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should be identical in shape so that the ratio v/EP ought to be independent of the concentration of ATP. We tested this prediction by parallel measurements of v and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K+, Rb+ or NH+4. In the absence of K+ or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical Km values (0.16 microM) so that v/EP remained independent of [ATP]. In the presence of either K+, Rb+ or NH+4, v and EP increased with [ATP] along the sum of two Michaelis-Menten equations. The biphasic response of v is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio v/EP increased with [ATP] from 19.8 to 40.1 s-1 along a hyperbola that was half-maximal at 9.5 microM. To preserve the validity of the current model it seems necessary to assume that ATP acts on the E1P <--> E2P transition and/or on the rate of hydrolysis of E2P. The latter possibility was ruled out. We also found that to fit the Albers-Post model to our data, the rate constant for K+ deocclussion from E2 has to be about 10-times higher than that reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts the experimental behavior of the Na(+)-ATPase activity but is unable to do this for the Na+/K(+)-ATPase activity, unless additional and yet unproved hypothesis are included.

The dephosphorylation reaction of the Ca(2+)-ATPase from plasma membranes.

The breakdown of phosphoenzyme (EP) of the Ca(2+)-ATPase from pig red blood cell membranes was studied at 37 degrees C by means of a rapid chemical quenching technique. When the enzyme was phosphorylated with [gamma-32P]ATP in media without added MgCl2, all the EP formed disappeared along two single exponential curves, a rapid one with k(app) = 90 +/- 10 s-1 and a slow one with k(app) = 0.7 +/- 0.3 s-1. The amount of EP involved in each reaction was close to 50% of the EP present at the beginning. Only EP of rapid breakdown could account for the steady-state hydrolysis of ATP observed under the same experimental conditions. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s-1) with K0.5 = 740 +/- 120 microM as if this reaction represented the decay of CaE1P, which donated its phosphate to water slowly in the forward direction and rapidly to ADP in the reverse direction of the cycle. Combination of Mg2+ with K0.5 = 26.3 +/- 5.0 microM at a single class of site in E1 before phosphorylation increased EP of rapid breakdown at the expense of ADP-sensitive EP so that, at nonlimiting concentrations of Mg2+ in the phosphorylation media, all EP decomposed at high rate. Rapid decomposition was observed even with enough CDTA to chelate most of the Mg2+ remaining from phosphorylation, suggesting that the role of Mg2+ during dephosphorylation was to accelerate the transition CaE1P-->CaE2P, preparing EP for hydrolysis. The combination of ATP at a single class of site with Km = 845 +/- 231 microM accelerated the hydrolysis of CaE2P. Calmodulin alone had no effects on dephosphorylation but enhanced acceleration of hydrolysis of CaE2P by ATP making the decay of EP under these conditions the fastest among those measured. Comparison of the rates of dephosphorylation of EP made in the presence of Mg2+ with those of steady-state Ca(2+)-ATPase activity with and without calmodulin showed that the transition CaE1P-->CaE2P and decomposition of CaE2P by hydrolysis are compatible with their role as obligatory intermediate reactions in the cycle of hydrolysis of ATP by the Ca(2+)-ATPase.

Low affinity acceleration of the phosphorylation reaction of the Na,K-ATPase by ATP.

The maximum rate of phosphorylation (rm) of a highly purified Na,K-ATPase from red outer medulla of pig kidney was measured at 25 degrees C as a function of ATP concentration in media with Mg2+, Na+, and no K+. When rm was plotted as a function of the concentration of ATP a biphasic response was observed with a hyperbolic component of high affinity (Km = 15.7 +/- 2.6 microM) and low velocity ((rm)max = 460 +/- 40 nmol of Pi/(mg of protein.s)) plus a parabolic component which showed no saturation up to 1000 microM ATP, concentration at which rm was 1768.1 +/- 429.6 nmol Pi/(mg protein.s) (mean +/- S.E.; n = 3). This low affinity effect of ATP on the rate of phosphorylation disappeared when the Na,K-ATPase underwent turnover in medium without K+ suggesting that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-6601), it required the enzyme to be at rest. This property of the Na,K-ATPase was not predicted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme (Er) functionally different from E1 or E2, which is able to bind three molecules of ATP, one with high and two with low affinity, and that after phosphorylation is converted into the phosphointermediates that are generally considered to participate in the reaction cycle described by Albers and Post.

Low affinity superphosphorylation of the Na,K-ATPase by ATP.

Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.

Does calmodulin regulate the affinity of the human red cell Ca2+ pump for ATP?

(1) We have reexamined the effects of calmodulin and of the calmodulin antagonist, compound 48/80 on the interaction of ATP at its low-affinity site in the Ca2(+)-ATPase from human red cells. (2) At variance with our earlier proposal (Biochim. Biophys. Acta (1985) 816, 379-386) calmodulin increased the maximum effect of ATP without changing the apparent affinity for ATP at the low-affinity site. Accordingly, ATP increased the maximum activation by calmodulin without altering the apparent affinity of the Ca2(+)-ATPase for calmodulin. (3) Confirming our previous observation (Biochim. Biophys. Acta (1985) 816, 379-386) compound 48/80 lowered the apparent affinity of the Ca2(+)-ATPase for ATP at the low-affinity site. This has to be attributed to a direct effect of this compound on the enzyme rather than to its effect as calmodulin antagonist.

Magnesium-ions accelerate the formation of the phosphoenzyme of the (Ca2+ + Mg2+)-activated ATPase from plasma membranes by acting on the phosphorylation reaction.

Magnesium ions in the reaction medium at 37 degrees C increased up to 222 s-1 the kapp for phosphorylation by ATP of the Ca2(+)-ATPase of pig red cell membranes. This effect was observed after partial proteolysis with trypsin which makes the enzyme behave like the E1 conformer during phosphorylation. These findings lead to the conclusion that Mg2+ increased the rate of phosphorylation of the Ca2(+)-ATPase by acting directly on this reaction. The apparent dissociation constant of Mg2+ for this effect was 44 microM whereas the apparent dissociation constant for Mg2+ to accelerate the shift E2----E1 between conformers measured on the intact enzyme was 50 microM. This suggests that Mg2+ accelerated both reactions from a single class of site.

The E2 in equilibrium E1 transition of the Ca2(+)-ATPase from plasma membranes studied by phosphorylation.

The relative abundance of the two conformers (E1 and E2) of the Ca2(+)-ATPase of plasma membranes and the rates of their interconversion were estimated measuring the initial velocity of phosphorylation of the Ca2(+)-ATPase in pig red cell membranes at 37 degrees C. This was based on the hypothesis that only E1 catalyzes phosphorylation from ATP. In the absence of ligands near 90% of the Ca2(+)-ATPase was in the E2 conformation. Ca2+ shifted the equilibrium toward E1. The K0.5 of Ca2+ for this effect was 15 microM, suggesting that it acted at the transport site. The conversion of E2 into E1 was slow (t1/2 = 911 s) while the conversion of E1 into E2 was faster (t1/2 less than or equal to 60 s). Mg2+ accelerated the E2----E1 reaction lowering its t1/2 to 0.25 s. In the presence of 4 mM Ca2+ t1/2 was 7.8 s, as if at this concentration to some extent Ca2+ replaced Mg2+ in accelerating the E2----E1 reaction. Results suggest that in intact membranes at 37 degrees C Ca2+ stabilized E1 and that the Ca2(+)-induced E2----E1 transition was strongly accelerated by Mg2+. Both cations were effective at near physiological concentrations and in the absence of other ligands like ATP or calmodulin that could also modify the reaction. After partial proteolysis with trypsin the Ca2(+)-ATPase behaved during phosphorylation as if it were E1.

Steady-state kinetic analysis of the Na+/K+-ATPase. The effects of adenosine 5'-[beta, gamma-methylene] triphosphate on substrate kinetics.

We studied the substrate kinetics of the Na+/K+-ATPase in media with adenosine 5'-[beta, gamma-methylene] triphosphate ([beta,gamma-CH2]ATP), an analog of ATP that is resistant to enzymatic hydrolysis. The aim was to analyze from the point of view of steady-state kinetics the mechanism that generates the biphasic response of the Na+/K+-ATPase to ATP. In the absence of K+, the analog acted as a dead-end inhibitor, Kt for this effect was 43.4 muM. In media with K+ and non-saturating concentrations of ATP, [beta, gamma-CH2]ATP stimulated ATPase. With high concentrations of [beta,gamma-CH2]ATP, the response of activity to the concentration of ATP changed from biphasic to hyperbolic. Comparison of these effects with the predictions of reaction mechanisms that display biphasic responses to the substrate showed that: (1) when this response is caused by two independent and non-interacting active sites, an analog of the substrate will not activate but may change the substrate curve from biphasic into hyperbolic, (ii) if there were negative interactions in affinity and positive interactions in reactivity between two active sites, an analog may activate and change the substrate curve from biphasic into hyperbolic, (iii) in models, such as that proposed for the Na+/K+-ATPase by Plesner et al. in 1981 (Blochim. Biophys. Acta 643, 483-494) in which biphasic kinetics is caused by the existence of two reaction cycles for a single active site, the analog will not activate, and (iv) the observed effects of the analog are compatible with models such as that of Albers-Post model and the more recent versions of the Plesner et al. model in which ATP apart from being the substrate is required to accelerate the rate-limiting step of the reaction.

Steady-state kinetic analysis of the Na+/K+-ATPase. The activation of ATP hydrolysis by cations.

We studied the interactions between pairs of cations during activation of the steady-state hydrolysis of ATP of the Na+/K+-ATPase. Non-linear regression was used to obtain empirical equations that describe quantitatively the behaviour of the system. The curve relating activity to Na+ concentration was describable by a Hill equation with nH = 2 and not by the more frequently used expression based on rapid-equilibrium binding of Na+ to three identical and non-interacting sites. At non-limiting concentrations of the other ligands, changes in the concentration of Na+ or of Mg2+ modified in the same proportion the maximum effects and the apparent affinities of K+, revealing the operation of either ping-pong or of ordered sequential mechanisms with irreversible steps separating the additions of each ligand. In contrast with this, changes in the concentration of Mg2+ altered only the maximum effect of Na+, indicating that a ternary complex between the cations and the enzyme has to be formed and that certain particular relations have to hold among the rate constants of the system. The interactions described in this paper, together with those previously reported, allowed us to derive a general equation that adequately predicted the response of the Na+/K+-ATPase to the concentration of any pair of ligands at non-limiting concentrations of the rest. Confrontation of this equation with computer simulations of the behaviour of the Albers-Post model shows that this model predicts the interactions in which K+ participates and perhaps also the interaction between Mg2+ and Na+, but seems unable to predict the interactions between pairs of ligands that do not include K+.

Steady-state kinetic analysis of the Na+/K+-ATPase. The inhibition by potassium and magnesium.

At micromolar ATP, low (< 5 mM) concentrations of K+ activate the Na+/K+-ATPase to an extent that is substantially reduced compared to that observed at more physiological concentrations of the nucleotide. At higher concentrations of K+, activation is replaced by partial inhibition. Inhibition is not due to the displacement of Na+ by K+, its main causes being a decrease of the Vm of the high-affinity component and the increase in the apparent Km of the low-affinity component of the substrate curve of the Na+/K+-ATPase. The apparent affinity for inhibition by K+ is highly dependent on Mg2+. In the presence of an excess K+, Mg2+ decreases towards zero the Vm of the high-affinity component and acts as a dead-end inhibitor of the low-affinity component of the substrate curve of the Na+/K+-ATPase. These results can be explained assuming that binding of an additional K+ to the E2 conformer of the Na+/K+-ATPase allows low-affinity binding of Mg2+ with the formation of a dead-end complex. In the case of Na+-ATPase activity and for concentrations of ATP within the range of the substrate curve of this activity (0-2.5 muM), Mg2+ in concentrations up to 60 mM has no effect on ATPase activity at high (100 mM) [Na+]. At lower [Na+], Mg2+ becomes a low-affinity inhibitor of Na+-ATPase. Inhibition follows a pattern that is different from inhibition of Na+/K+-ATPase activity and is consistent with a mechanism in which Mg2+ acts both as a dead-end and as product inhibitor.

Pre-steady-state phosphorylation of the human red cell Ca2+-ATPase.

The pre-steady-state kinetics of phosphorylation of the Ca2+-ATPase by ATP was studied at 37 degrees C and in intact red cell membranes to approach physiological conditions. ATP and Ca2+ activate with K0.5 of 4.9 and 26.4 microM, respectively. Preincubation with Ca2+ did not change the K0.5 for ATP. Preincubation with ATP did not alter the initial velocity of phosphorylation suggesting that binding of ATP was not rate-limiting. Mg2+ added at the start of the reaction increased the initial rate of phosphorylation from 4 to 8 pmol/mg/s. With 30 microM Ca2+, the K0.5 for Mg2+ was 60 microM. Mg2+ and Ca2+ added together beforehand accelerated phosphorylation to 70 pmol/mg/s. Phosphorylation of calmodulin-bound membranes was the fastest (280 pmol/mg/s), and its time course showed a neat overshoot before steady state. The results suggest that either preincubation with Ca2+ plus Mg2+ or calmodulin accelerated phosphorylation shifting toward E1 the equilibrium between the E1 and E2 conformers of the enzyme. K+ had no effect on the initial rate of phosphorylation and lowered by 40% the steady-state level of phosphoenzyme in the absence of Mg2+. Phosphorylation is not rate-limiting for the overall reaction since its initial rate was always higher than ATPase activity. In the absence of K+, the turnover of the phosphoenzyme was 2000 min-1, which is close to the values for other transport ATPases.

Comparison between plasma membrane Ca2+ and Na,K-ATPases: short review.

1. This paper is a short review of the comparative biochemistry of the Na,K-ATPase and Ca2+-ATPase of plasma membranes. 2. The two ATPases share the same biphasic activation by ATP. Ca2+-ATPase activation by ATP is strongly affected by calmodulin. 3. The possibility of Mg2+ occlusion is proposed in connection with low-affinity activation by ATP. 4. Both ATPases are activated by alkaline earth metal ions and display phosphatase activity toward p-nitrophenylphosphate for which Ca2+-ATPase is strongly dependent on K+ and regulated by calmodulin. 5. The requirements for ligands of the phosphatase activity of both ATPases are strikingly similar except for the effect of calmodulin. 6. Both ATPases are inhibited by vanadate and for both the effect of vanadate is modulated by Mg2+ and K+ in the same way. 7. These similarities indicate that, although Ca2+-ATPase and Na,K-ATPase are different enzymes, their mechanisms of action may have more features in common than previously thought.

Comparasion between plasma membrane Ca2 + and Na, K-ATPases: short review

This paper is a short review of the comparative biochemistry of the Na, K-ATPase and Ca2+ -ATPase of plasma membranes. The two ATPases share the same biphasic activation by ATP. Ca2+ - ATPase activation by ATP is strongly affected by calmodulin. The possibility of Mg2+ occlusion is proposed in connection with low-affinity activation by ATP. Both ATPase are activated by alkaline earth metal ions and display phosphatase activity toward p-nitrophenylphosphat for which Ca2 + - ATPase is strongly dependent on K + and regulated by calmodulin. The requirements for ligands of the phosphatase activity of both ATPase are strikingly similar except for the effect of calmodulin. Both ATPases are inhibited by vanadat and for both the effect of vanadate is modulated by Mg2+ and K + in the same way. These similarities indicate that, although Ca2 + - ATPase and Na, K-ATPase are different enzymes, their mechanisms of action may have more feactures in common than previously thought

Differential effects of compound 48/80 on the ATPase and phosphatase activities of the Ca2+ pump of red cells.

The calmodulin antagonist compound 48/80 inhibits the phosphatase activity of the Ca2+-ATPase lowering its maximum velocity and leaving unaltered its apparent affinity for the substrate regardless on whether phosphatase activity is elicited by Ca2+ plus ATP or by calmodulin. Compound 48/80 has no effect on the Ki for ATP as inhibitor of the phosphatase. These results contrast sharply with the large increase that compound 48/80 induces in the apparent affinity of the regulatory site for the nucleotide of the Ca2+-ATPase and suggest that the active site for phosphatase activity is different from the regulatory site for ATP of the Ca2+-ATPase.