The Sodium- and Potassium-Activated Adenosinetriphosphatase System

Schwartz, Arnold; Nagano, Kei; Nakao, Makoto; Lindenmayer, George E.; Allen, Julius C.; Matsui, Hideo

doi:10.1007/978-1-4615-7425-5_12

Cited by 129 publications

(101 citation statements)

References 26 publications

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“…Protein concentration was assessed by the Lowry method, using bovine serum albumin as a standard. Specific ATPase activity was determined by the pyruvate kinase/lactate dehydrogenase assay [34]. The specific activity was in the range of 1900 to 2300 M P i per mg protein and h at 37°C.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…The most active fractions of the final density gradient separation had a protein content of 2-3 mg/ml. The enzymatic activity was determined by the pyruvate kinase/lactate dehydrogenase linked-assay (Schwartz et al, 1971) in a buffer containing 2 M of free Ca 2+ . Background enzymatic activity of the isolated preparation was obtained by either quantitative removal of the free Ca 2+ by 5 mM EGTA or addition of 1 M tharpsigargin.…”

Section: Methodsmentioning

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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“…Na+ ions) also favour and stabilize the binding of cardioactive steriods to the receptor site, whereas ligands leading to the dephosphorylated state (e.g. K+ ions) decrease the binding rate of the cardioactive steriods (Schwartz et al, 1975;Repke & Schonfeld, 1984). The interaction is mutual, since occupancy of the extracellular receptor site by a cardioactive steroid immediately and drastically inhibits the K+-promoted dephosphorylation step.…”

Section: Receptor Topographymentioning

confidence: 99%

“…This is a typical ligandinduced conversion. For detailed descriptions of ligand-induced receptor conversion consult, for instance, Schwartz et al (1975), Erdmann (1984), Hansen (1984), , Repke & Schonfeld (1984).…”

Section: The Ouabain-resistance Genementioning

confidence: 99%