Structural basis for E1–E2 conformational transitions in Na, K-pump and Ca-pump proteins

Jørgensen, Peter Leth; Andersen, Jens Peter

doi:10.1007/bf01870942

Cited by 334 publications

(186 citation statements)

References 194 publications

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“…Specific ATPase activity was measured by the pyruvate kinase/lactate dehydrogenase assay (18), and the protein concentration was determined by the Lowry method (19), using bovine serum albumin as a standard. For the calculation of the molar protein concentration, a molecular weight of an R unit of the Na + ,K + -ATPase of 147,000 g mol -1 (20) was assumed. The specific activity of the Na + ,K + -ATPase preparations used was in the range of 1900-2040 µmol P i /h per mg of protein at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

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

2007

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“…Assay at 25 M ATP, 130 mM Na ϩ , and 2 mM K ϩ were done in a recording spectrophotometer as described before (20 (2). The high affinity inhibitor, ouabain, binds to the E 2 P form.…”

Section: Methodsmentioning

confidence: 99%

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

Pedersen

Jørgensen

2000

Journal of Biological Chemistry

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The segment 708 TGDGVNDSPALKK 720 in the ␣-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg 2؉ 710 3 Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl ؉ 2-to 3-fold, suggesting a role in transmission of K ؉ stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

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“…There are controversies about whether the enzyme exists as a protomer (aß) (21)(22)(23)37) or as a diprotomer (aß) 2 involving a balance between protomer-diprotomer, or even in the form of oligomers (aß) n (24,38).…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,KATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8 -solubilized and purified Na,KATPase by 99, 81 and 98.5%, respectively. We have shown that Na,KATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.

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Structural basis for E1–E2 conformational transitions in Na, K-pump and Ca-pump proteins

Cited by 334 publications

References 194 publications

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

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

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

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Structural basis for E1–E2 conformational transitions in Na, K-pump and Ca-pump proteins

Cited by 334 publications

References 194 publications

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

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

Importance of Conserved α-Subunit Segment709GDGVND for Mg2+ Binding, Phosphorylation, and Energy Transduction in Na,K-ATPase

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

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

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