Rolando C. Rossi

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.

Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.

THE INTERACTION OF K+ Na+, Mg2+, AND ATP WITH THE (Na, K)-ATPase*

Since the initial observations by Post and coworkers,’ a large amount of information has accumulated indicating that hydrolysis of ATP by the (Na,K)ATPase proceeds through a series of steps involving the formation and hydrolysis of pho~phoenzymes.~-~ This information has led to a detailed reaction scheme for the hydrolysis of ATP and for the associated active transport of Na+ and K+.2.3 The main experimental support for the currently accepted reaction scheme comes from studies on partial reactions. Few attempts have been made to see to what extent this scheme is able to predict quantitatively the steady-state kinetic behavior of the (Na,K)-ATPase. Studies of this kind are important since agreement between theoretical predictions and experimental results is one of the necessary requirements for the validity of any reaction scheme. We will report here experiments on the interaction of ATP, K+, Na+, and M&+ with the (Na,K)-ATPase during steady-state ATP hydrolysis. We will try to show that comparison of the experimental results with theoretical kinetic equations seems to indicate that the behavior of the system is in some respects difficult to reconcile with the predictions of the currently held view about the role of Na+, K+, M g + , and ATP during hydrolysis of ATP by the (Na,K]-ATPase. The experiments reported here were performed using a partially purified (Na,K)-ATPase isolated from the outer medulla of dog kidney following Jdrgensen’s procedure “C.”4 ATPase activity was estimated from the initial rate of release of 32P (Pi) from [y-3ZP]ATP.5 Incubations were carried out at 37OC, adjusting the length of incubation and the concentration of enzyme so that no more than 10% of the ATP underwent enzymatic hydrolysis. In all media, pH was kept at 7.4 with 20 mM imidazol-HC1 and total salt concentration at 150 mM by mixing adequate amounts of NaCl, KCI, and choline C1. All media contained 1% (w/v] of bovine serum albumin.

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.

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca2+-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca2+ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μm, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of LaIII (inducing accumulation of phosphoenzyme in the E1P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E1P-phosphorylated intermediate of the PMCA.

Binding of a Single Rb+ Increases Na+/K+-ATPase, Activating Dephosphorylation without Stoichiometric Occlusion

We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 μm ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained.

Conformational changes produced by ATP binding to the plasma membrane calcium pump

Background: The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes. Results: We identified different conformations after the association of Ca2+, ATP, and vanadate to PMCA. Conclusion: PMCA forms a stable complex with Ca2+ and vanadate; ATP can bind to all pump conformations. Significance: This study found a new intermediate in the PMCA reaction cycle; all of the intermediates interact with ATP. The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Dynamic lipid-protein stoichiometry on E1 and E2 conformations of the Na+/K+ -ATPase.

Annular lipid-protein stoichiometry in native pig kidney Na+/K+ -ATPase preparation was studied by [125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in E2, i.e., the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E2 conformation.

Rb(+) occlusion stabilized by vanadate in gastric H(+)/K(+)-ATPase at 25°C.

Despite its similarity with the Na(+)/K(+)-ATPase, it has not been possible so far to isolate a K(+)-occluded state in the H(+)/K(+)-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb(+) (as surrogate for K(+)) in H(+)/K(+)-ATPase from gastric vesicles at 25°C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb(+) ions from the intravesicular medium than C(12)E(8) (a non-ionic detergent). In the presence of vanadate and Mg(2+), the time course of [(86)Rb]Rb(+) uptake displayed a fast phase due to Rb(+) occlusion. The specific inhibitor of the H(+)/K(+)-ATPase SCH28080 significantly reduces the amount of Rb(+) occluded in the vanadate-H(+)/K(+)-ATPase complex. Occluded Rb(+) varies with [Rb(+)] according to a hyperbolic function with K(0.5)=0.29±0.06mM. The complex between the Rb(+)-occluded state and vanadate proved to be very stable even after removal of free Mg(2+) with EDTA. Our results yield a stoichiometry lower than one occluded Rb(+) per phosphorylation site, which might be explained assuming that, unlike for the Na(+)/K(+)-ATPase, Mg(2+)-vanadate is unable to recruit all the Rb(+)-bound to the Rb(+)-occluded form of the Rb(+)-vanadate-H(+)/K(+)-ATPase complex.

Alternative cycling modes of the Na(+)/K(+)-ATPase in the presence of either Na(+) or Rb(+).

A comprehensive study of the interaction between Na(+) and K(+) with the Na(+)/K(+)-ATPase requires dissecting the incidence of alternative cycling modes on activity measurements in which one or both of these cations are absent. With this aim, we used membrane fragments containing pig-kidney Na(+)/K(+)-ATPase to perform measurements, at 25°C and pH=7.4, of ATPase activity and steady-state levels of (i) intermediates containing occluded Rb(+) at different [Rb(+)] in media lacking Na(+), and (ii) phosphorylated intermediates at different [Na(+)] in media lacking Rb(+). Most relevant results are: (1) Rb(+) can be occluded through an ATPasic cycling mode that takes place in the absence of Na(+) ions, (2) the kinetic behavior of the phosphoenzyme formed by ATP in the absence of Na(+) is different from the one that is formed with Na(+), and (3) binding of Na(+) to transport sites during catalysis is not at random unless rapid equilibrium holds.