Kinetics of Na+-Dependent Conformational Changes of Rabbit Kidney Na+,K+-ATPase

Clarke, Ronald J.; Kane, David; Apell, Hans-Jürgen; Roudná, Milena; Bamberg, Ernst

doi:10.1016/s0006-3495(98)74052-4

Cited by 43 publications

(94 citation statements)

References 59 publications

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“…The ATP dissociation constant for the E 2 state of the enzyme was chosen to have a value of 71 µM, consistent with literature data (7). Both the predicted time course of the fluorescence change and its total amplitude as a function of ATP concentration were calculated.…”

Section: Resultsmentioning

confidence: 99%

“…This was first demonstrated by Karlish and Yates (2), who measured the rate of the E 2 f E 1 conformational transition, which occurs simultaneously with K + release, via the stopped-flow technique, utilizing the change in the enzyme's tryptophan fluorescence as the means of detection. This stimulation of the E 2 f E 1 transition by ATP has since been confirmed by a number of groups using a variety of experimental approaches (3)(4)(5)(6)(7)(8). The dissociation constant of ATP for the E 2 conformation has been estimated to be 71 µM (7), 143 µM (6), 196 µM (4), 277-303 µM (8), 300 µM (3), and 450 µM (2).…”

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confidence: 75%

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Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

2007

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The kinetics of the E 2 f E 1 conformational change of unphosphorylated Na + ,K + -ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E 2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na 2 ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of g50 µM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E 2 :E 2 f E 2 :E 1 f E 1 :E 1 . In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E 2 :E 2 f E 2 :E 1 and E 2 :E 1 f E 1 :E 1 transitions were found to be 10.4 ((1.0) and 0.49 ((0.02) s -1 , respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 ((0.3) s -1 . It was found that ATP acting at a high affinity site (K d ≈ 0.25 µM), stimulated the reverse reaction, E 1 ATP f E 2 ATP, in addition to its known allosteric low affinity (K d ≈ 71 µM) stimulation of the forward reaction, E 2 ATP f E 1 ATP. Na + ,K + -ATPase 1 was the first ion pump to be discovered (1), and it is one of the most fundamentally important enzymes of animal physiology. The electrochemical potential gradient for Na + ions, which the enzyme maintains, is used as the driving force for numerous secondary transport systems. Examples include the Na + channels in the nerve, which allow the action potential to be produced, the Na + -glucose cotransporter, which is responsible for glucose uptake in intestinal cells, and the Na + /Ca 2+ exchanger in the heart, which plays an important role in muscle relaxation. Na + ,K + -ATPase, furthermore, contributes to the osmotic regulation of cell volume and is a major determinant of body temperature. For all of these functions, the enzyme derives its energy from the hydrolysis of ATP, which is coupled to Na + transport.For many years, it has been known that ATP also has an allosteric effect on the enzyme. Under physiological conditions, ATP binds to the E 2 (K + ) 2 conformation of the enzyme and accelerates the release of K + ions to the cytoplasm without undergoing any hydrolysis. This was first demonstrated by Karlish and Yates (2), who measured the rate of the E 2 f E 1 conformational transition, which occurs simultaneously with K + release, via the stopped-flow technique, utilizing the change in the enzyme's tryptophan fluorescence as the means of detection. This stimulation of the E 2 f E 1 transition by ATP has since been confirmed...

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Section: Resultsmentioning

confidence: 99%

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confidence: 75%

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

2007

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“…The electrogenic contributions of such reaction steps may be described in terms of dielectric coefficients, and the sum of the dielectric coefficients of all reaction steps in the pump cycle equals the net charges translocated across the membrane (Läuger & Apell, 1986, Läuger, 1991. Styryl dyes, especially RH421, were used with great success to investigate Na,K-ATPase-containing membrane preparations (Klodos & Forbush, 1988, Pratap et al, 1993Heyse et al, 1994, Fedosova, Cornelius & Klodos, 1995Kane et al, 1995;Apell et al, 1996;Clarke et al, 1998). Significant fluorescence changes have been observed in all partial reactions which contain electrogenic reaction steps.…”

Section: Discussionmentioning

confidence: 99%

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Butscher

Roudná

Apell

1999

Journal of Membrane Biology

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Abstract.A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/ release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca 2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca 2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca 2+ ions in state E 1 with an apparent half-saturation concentration, K M of 600 nM. In state P-E 2 two K M values, 5 M and 2.2 mM, were determined and are in fair agreement with published data. From Ca 2+ titrations in buffers with various pH and from pH titrations in P-E 2 , it could be demonstrated that H + binding is electrogenic and that Ca 2+ and H + compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E 1 with unoccupied ion sites, independent of the buffer composition.

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“…During the recent years the transport cycle [1,3,27] has been broken down into a series of defined reaction steps and their kinetic properties were investigated and characterized [5,10,11,15,17,40]. In the case of the sodium transport branch the reaction sequence, 3 Na + cyt + E 1 → Na 3 E 1 → ͑Na 3 ͒E 1 -P → P-E 2 ͑Na 3 ͒ → P-E 2 ͑Na 2 ͒ + Na + → P-E 2 + Na + ext , is able to explain all experimental data available.…”

Section: Introductionmentioning

confidence: 99%

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

Schneeberger

Apell

2001

Journal of Membrane Biology

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Abstract.In the E 1 state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di-and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br 2 -Titu 3+ is bound with the highest affinity (< M), among the tested divalent cations Ca 2+ binds the strongest, and Na + binds with about the same equilibrium dissociation constant as Mg 2+ (∼0.8 mM). For alkali ions it exhibits binding affinities following the order of Rb + Ӎ K + > Na + > Cs + > Li + . The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na + ions the equilibrium dissociation constant is < 0.01 mM. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na + , no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mM.

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Kinetics of Na+-Dependent Conformational Changes of Rabbit Kidney Na+,K+-ATPase

Cited by 43 publications

References 59 publications

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

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Kinetics of Na+-Dependent Conformational Changes of Rabbit Kidney Na+,K+-ATPase

Cited by 43 publications

References 59 publications

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

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Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics