Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover.

Campos, Marta; Beaugé, Luis

doi:10.1016/s0021-9258(17)32413-4

Cited by 28 publications

(15 citation statements)

References 36 publications

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“…An observation in favor this idea is the fact that the chymotrypsin-treated Na + ,K + -ATPase cannot be phosphorylated from P i even in the presence of ouabain (). As this P i incorporation occurs only in the E 2 state ( , ), it may very well be that this enzyme does not go into E 2 . (b) In the presence of phosphorylation from ATP ( , ), and the nonnucleotide fueling substrate acetyl phosphate (), the enzyme occludes Na + ; in both instances Na + is released in the presence of ADP, a typical E 1 P behavior.…”

Section: Discussionmentioning

confidence: 99%

“…NaCl and KCl were of spectrometric grade. Vanadium-free ATP and ADP were from Boehringer Mannheim; the other chemicals, of reagent grade, were obtained from Sigma Chemical Co. Inorganic [ 32 Counting. Radioactivity assays were performed in a Beckman liquid scintillation counter using a toluene-based scintillation fluid; counting times were long enough to obtain standard errors of about 1%.…”

Section: Methodsmentioning

confidence: 99%

“…The total number of phosphorylating sites was measured separately as indicated above. The estimation of f 1 and f 2 was done using two syringes following the timedependent EP formation by varying the concentration of [γ- 32 P]ATP at constant but different ionized [Mg 2+ ]. The elected [ATP] were 0.75, 1.5, 3, 5, 10, 25, 50, and 100 µM.…”

Section: (Iii) Simultaneous Estimation Of the On Rate Of Atp Binding ...mentioning

confidence: 99%

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ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase: Dephosphorylation by ADP of the E₁P Enzyme Form

Campos

Beaugé

1997

Biochemistry

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We studied the effects of Mg2+ and of ADP and other nucleoside diphosphates on the dephosphorylation of the E1P form of the partially purified pig kidney Na+,K+-ATPase at 20-22 degrees C. We report for the first time the rate of the reversal of ATP phosphorylation. The experiments were done on enzyme subjected to controlled chymotrypsin digestion consisting of a homogenous population of a truncated catalytic subunit. Under this condition the whole cycle is E1 <-- (f1.ATP, b1) --> E1ATP <-- (f2, b2) --> E1P.ADP <-- (fd, bd.ADP) --> E1P-(f3) --> E1. The values of f1, b1, f2, and f3 were independently estimated in the absence of ADP; those of fd, bd, and b2 were obtained from the fit of ADP-dependent dephosphorylation data to the differential equation set. When f2 = 0 or b1 is very large, the model predicts that dephosphorylation by ADP gives a single exponential; in all other cases it predicts a biphasic dephosphorylation in a semilogarithmic plot. The fast phase is governed by b2.ADP and the slow one by b1. This was experimentally verified. Also, ADP stimulates E1P breakdown without release of Pi, thus leading to ATP synthesis. The data indicate that the true substrate for ATP synthesis is free ADP, while Mg2+ inhibits mainly by a reduction in the free [ADP]; in addition, E1P has a very low affinity for MgADP. The nucleotide structure is also very important; all ADP analogues tested were much less effective than ADP due to a reduced affinity for the E1P and a poor capacity to reverse phosphorylation.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: (Iii) Simultaneous Estimation Of the On Rate Of Atp Binding ...mentioning

confidence: 99%

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ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase: Dephosphorylation by ADP of the E₁P Enzyme Form

Campos

Beaugé

1997

Biochemistry

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“…Here we extend these analyses of the Na,K-ATPase through investigation of the phosphorylation of the enzyme by P i , the so-called "back-door" phosphorylation, that has previously been studied mainly by 32 P-phosphorylation methods. It has been shown that P-E 2 formation from P i can occur provided Na + is absent (Campos & Beauge ´, 1994;Cornelius, 1995;Post et al, 1975). Phosphorylation from P i or ATP results in chemically identical phosphoenzymes (Bonting et al, 1979;Berberia ´n & Beauge ´, 1991).…”

mentioning

confidence: 99%

Kinetics of the Phosphorylation of Na,K-ATPase by Inorganic Phosphate Detected by a Fluorescence Method

et al. 1996

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Phosphorylation by P i of the Na,K-ATPase from rabbit kidney in the absence of Na + ions but in the presence of Mg 2+ ions has been studied. In the absence of K + ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the two states were monitored. Transient kinetic studies of phosphorylation were initiated by manual addition of P i or by photochemical release of P i from 1-(2-nitrophenyl)ethyl phosphate (caged P i ) using laser flash photolysis at 308 nm. Equilibrium studies of phosphorylation showed that the apparent K m for P i was 23.0 ( 0.3 µM (mean ( sem) at pH 7.1 and 21°C. The dye fluorescence increased in a biphasic manner on addition of 500 µM P i to the enzyme: a rapid phase (t 1/2 < 1 s) and a slower exponential phase at 0.059 ( 0.003 s -1 . The rate of the rapid phase was studied by fast concentration-jump experiments and exhibited first-order kinetics in P i up to 60 µM. Fluorescence records vs time were exponential, and a plot of the rate constant versus [P i ] had a slope of 1.47 × 10 5 M -1 s -1 and ordinate ([P i ] ) 0) intercept of 3.1 s -1 . Addition of 50 mM NaCl to the phosphorylated enzyme induced an exponential decay in the dye fluorescence from which a rate constant of 0.10 ( 0.005 s -1 was determined. These data were interpreted in terms of transformations between conformational states E 1 and E 2 , and the phosphorylated state P-E 2 defined in the Post-Albers mechanism of the Na,K-ATPase [Läuger, P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA] ass -1 , and k -2 ) 3.1 s -1 . The RH421 fluorescence of state P-E 2 was studied over the pH range 6-8.5. Fluorescence was greatest at pH 8.5 and lowest at pH 6.0 in a simple binding isotherm with pK 7.5. The apparent K m for P i rose cooperatively with increasing pH (pK a 8.6 and a Hill coefficient of 2). Therefore in the absence of monovalent metal ions, occupation of the cation (K + ) binding sites by protons promotes phosphorylation by P i .

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“…The rate constants of the reactions described in tables 1 and 2 were extracted from published data (table 4). The constants of reaction 1 were extracted from an experimental report where the authors investigated the pump cycle when only Na + is transported [12]. The constants of reactions 2 and 3 were based on values obtained from a detailed description of Na + and K + binding to the Na/K-ATPase during the complete transport cycle [13].…”

Section: P10mentioning

confidence: 99%

Hypothesized diprotomeric enzyme complex supported by stochastic modelling of palytoxin-induced Na/K pump channels

et al. 2018

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The sodium–potassium pump (Na+/K+ pump) is crucial for cell physiology. Despite great advances in the understanding of this ionic pumping system, its mechanism is not completely understood. We propose the use of a statistical model checker to investigate palytoxin (PTX)-induced Na+/K+ pump channels. We modelled a system of reactions representing transitions between the conformational substates of the channel with parameters, concentrations of the substates and reaction rates extracted from simulations reported in the literature, based on electrophysiological recordings in a whole-cell configuration. The model was implemented using the UPPAAL-SMC platform. Comparing simulations and probabilistic queries from stochastic system semantics with experimental data, it was possible to propose additional reactions to reproduce the single-channel dynamic. The probabilistic analyses and simulations suggest that the PTX-induced Na+/K+ pump channel functions as a diprotomeric complex in which protein–protein interactions increase the affinity of the Na+/K+ pump for PTX.

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Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover.

Cited by 28 publications

References 36 publications

ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase: Dephosphorylation by ADP of the E₁P Enzyme Form

ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase: Dephosphorylation by ADP of the E₁P Enzyme Form

Kinetics of the Phosphorylation of Na,K-ATPase by Inorganic Phosphate Detected by a Fluorescence Method

Hypothesized diprotomeric enzyme complex supported by stochastic modelling of palytoxin-induced Na/K pump channels

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Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover.

Cited by 28 publications

References 36 publications

ATP−ADP Exchange Reaction Catalyzed by Na+,K+-ATPase: Dephosphorylation by ADP of the E1P Enzyme Form

ATP−ADP Exchange Reaction Catalyzed by Na+,K+-ATPase: Dephosphorylation by ADP of the E1P Enzyme Form

Kinetics of the Phosphorylation of Na,K-ATPase by Inorganic Phosphate Detected by a Fluorescence Method

Hypothesized diprotomeric enzyme complex supported by stochastic modelling of palytoxin-induced Na/K pump channels

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ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase: Dephosphorylation by ADP of the E₁P Enzyme Form

ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase: Dephosphorylation by ADP of the E₁P Enzyme Form