Bioinformatic Study of Possible Acute Regulation of Acid Secretion in the Stomach.

The gastric H+,K+-ATPase is an integral membrane protein which derives energy from the hydrolysis of ATP to transport H+ ions from the parietal cells of the gastric mucosa into the stomach in exchange for K+ ions. It is responsible for the acidic environment of the stomach, which is essential for digestion. Acid secretion is regulated by the recruitment of the H+,K+-ATPase from intracellular stores into the plasma membrane on the ingestion of food. The similar amino acid sequences of the lysine-rich N-termini α-subunits of the H+,K+- and Na+,K+-ATPases, suggests similar acute regulation mechanisms, specifically, an electrostatic switch mechanism involving an interaction of the N-terminal tail with the surface of the surrounding membrane and a modulation of the interaction via regulatory phosphorylation by protein kinases. From a consideration of sequence alignment of the H+,K+-ATPase and an analysis of its coevolution with protein kinase C and kinases of the Src family, the evidence points towards a phosphorylation of tyrosine-7 of the N-terminus by either Lck or Yes in all vertebrates except cartilaginous fish. The results obtained will guide and focus future experimental research.

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport.

The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

Exploring the conformational transition between the fully folded and locally unfolded substates of Escherichia coli thiol peroxidase.

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.

Steady-state analysis of enzymes with non-Michaelis-Menten kinetics: The transport mechanism of Na+/K+-ATPase.

Procedures to define kinetic mechanisms from catalytic activity measurements that obey the Michaelis-Menten equation are well established. In contrast, analytical tools for enzymes displaying non-Michaelis-Menten kinetics are underdeveloped, and transient-state measurements, when feasible, are therefore preferred in kinetic studies. Of note, transient-state determinations evaluate only partial reactions, and these might not participate in the reaction cycle. Here, we provide a general procedure to characterize kinetic mechanisms from steady-state determinations. We described non-Michaelis-Menten kinetics with equations containing parameters equivalent to kcat and Km and modeled the underlying mechanism by an approach similar to that used under Michaelis-Menten kinetics. The procedure enabled us to evaluate whether Na+/K+-ATPase uses the same sites to alternatively transport Na+ and K+ This ping-pong mechanism is supported by transient-state studies but contradicted to date by steady-state analyses claiming that the release of one cationic species as product requires the binding of the other (ternary-complex mechanism). To derive robust conclusions about the Na+/K+-ATPase transport mechanism, we did not rely on ATPase activity measurements alone. During the catalytic cycle, the transported cations become transitorily occluded (i.e. trapped within the enzyme). We employed radioactive isotopes to quantify occluded cations under steady-state conditions. We replaced K+ with Rb+ because 42K+ has a short half-life, and previous studies showed that K+- and Rb+-occluded reaction intermediates are similar. We derived conclusions regarding the rate of Rb+ deocclusion that were verified by direct measurements. Our results validated the ping-pong mechanism and proved that Rb+ deocclusion is accelerated when Na+ binds to an allosteric, nonspecific site, leading to a 2-fold increase in ATPase activity.

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb(+).

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K2)Mg-MgF4, an E2∙Pi-like state capable to occlude K(+) (or Rb(+)). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb(+) with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1-E2 conformational transition and the kinetics of Rb(+) exchange between the medium and the E2(Rb2)Mg-MgF4 state. Our results show that both in the absence and in the presence of Rb(+), simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg-MgF4 complex, that is unable to shift to E1 upon addition of Na(+). The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg-MgF4 formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb(+) caused by Mg(2+). Na(+)-induced release of Rb(+) from E2(Rb2)Mg-MgF4 occurs at the same rate as from E2(Rb2) but is insensitive to ADP. The rate of Rb(+) occlusion into the E2Mg-MgF4 state is 5-8 times lower than that described for the E2Mg-vanadate complex. Since the E2Mg-MgF4 and E2Mg-vanadate complexes represent different intermediates in the E2-P→E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.

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.

E2→E1 transition and Rb(+) release induced by Na(+) in the Na(+)/K(+)-ATPase. Vanadate as a tool to investigate the interaction between Rb(+) and E2.

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).

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 (ß subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (ß 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.

Eosin fluorescence changes during Rb+ occlusion in the Na+/K(+)-ATPase.

We used suspensions of partially purified Na(+)/K(+)-ATPase from pig kidney to compare the effects of Rb(+), as a K(+) congener, on the time course and on the equilibrium values of eosin fluorescence and of Rb(+) occlusion. Both sets of data were collected under identical conditions in the same enzyme preparations. The incubation media lacked ATP so that all changes led to an equilibrium distribution between enzyme conformers with and without bound eosin and with and without bound or occluded Rb(+). Results showed that as Rb(+) concentration was increased, the equilibrium value of fluorescence decreased and occlusion increased along rectangular hyperbolas with similar half-maximal values. The time courses of attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolution of our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functions of time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the time course than in the case of occlusion. Comparison between experimental and simulated results suggests that fluorescence changes express a process that is coupled to Rb(+) occlusion but that is completed before occlusion reaches equilibrium.

Quantitative analysis of the interaction between the fluorescent probe eosin and the Na+/K+-ATPase studied through Rb+ occlusion.

We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K(+)-congener Rb(+) in the Na(+)/K(+)-ATPase. Eosin decreases the equilibrium levels of occluded Rb(+), this effect being fully attributable to a decrease in the apparent affinity of the enzyme for Rb(+) since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na(+)/K(+)-ATPase, both to the Rb(+)-free and to the Rb(+)-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb(+) occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that, like ATP, it increases the velocity of deocclusion of Rb(+). Interactions between eosin and ATP on Rb(+)-release experiments seem to indicate that eosin binds to the low-affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.

The Occlusion of Rb(+) in the Na(+)/K(+)-ATPase. I. The identity of occluded states formed by the physiological or the direct routes: occlusion/deocclusion kinetics through the direct route.

Occlusion of K(+) or its congeners in the Na(+)/K(+)-ATPase occurs after K(+)-dependent dephosphorylation (physiological route) or in media lacking ATP and Na(+) (direct route). The effects of P(i) or ATP on the kinetics of deocclusion of the K(+)-congener Rb(+) formed by each of the above mentioned routes was independent of the route of occlusion, which suggests that both routes lead to the same enzyme intermediate. The time course of occlusion via the direct route can be described by the sum of two exponential functions plus a small component of very high velocity. At equilibrium, occluded Rb(+) is a hyperbolic function of free [Rb(+)] suggesting that the direct route results in enzyme states holding either one or two occluded Rb(+). Release of occluded Rb(+) follows the sum of two decreasing exponential functions of time, corresponding to two phases with similar sizes. These phases are not caused by independent physical compartments. The rate constant of one of the phases is reduced up to 30 times by free Rb(+). When Rb(+) is the only pump ligand, the kinetics of occlusion and deocclusion through the direct route are consistent with an ordered-sequential process with additional independent step(s) interposed between the uptake or the release of each occluded Rb(+).