Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla

Hayashi, Yutaro; Homareda, Haruo; Kimimura, Mamiko

doi:10.1016/0006-291x(77)91052-x

Cited by 41 publications

(12 citation statements)

References 13 publications

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“…Yet, though Hastings & Skou (1980) working with Na, K-ATPase from the salt gland of the spiny dogfish, found that about five potassium ions could be bound per phosphorylation site, and Jensen & Ottolenghi (1982) working with Na, K-ATPase from pig kidney outer medulla, found a figure ofthree rubidium ions per ouabain-binding site, most workers have found figures near two. Thus in Na, K-ATPase from dog kidney outer medulla, Matsui, Hayashi, Homareda & Kimimura (1977) found that 1-7 potassium ions (displaceable by ouabain) were bound per ouabain-binding site; Matsui, Hayashi, Homareda & Taguchi (1982) found that 1-9 potassium ions (displaceable by ouabain) were bound per ATP-binding site; and Cantley, Cantley & Josephson (1978) found that about two rubidium ions were bound per vanadate-binding site. In Na, K-ATPase from pig kidney outer medulla, Yamaguchi & Tonomura (1979, 1980 and J0rgensen (1982) found that close to two rubidium ions were bound per phosphorylation site.…”

Section: Stoichiometrymentioning

confidence: 99%

Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Glynn

Richards

1982

The Journal of Physiology

155

113

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SUMMARY1. The occlusion of rubidium ions by Na, K-ATPase has been investigated by suspending enzyme prepared from pig kidney outer medulla in media containing low concentrations of 86Rb, forcing the suspensions rapidly through small columns of cation-exchange resin, and measuring the amounts of radioactivity emerging from the columns.2. When the suspension media contained 2 mM-ATP or ADP, or 15 mm-NaCl, the amounts of radioactivity emerging from the columns were greatly (and similarly) reduced, presumably because both nucleotides and sodium ions stabilized the enzyme in the E1 form. (See p. 19 for definition of E1 and E2). The extra radioactivity carried through the columns when nucleotides and sodium were absent was taken as a measure ofthe amount of rubidium occluded within the enzyme (in the E2 form) when it emerged from the resin.3. By varying the flow rate, and therefore the time spent by the enzyme on the resin, and relating this to the amount of radioactivity emerging from the columns, we have been able to estimate the rate constant for the conformational change (E2 -+ E1) that allows the occluded rubidium ions to escape. At 20 'C, and in the absence of nucleotides, it is about 0-1 s-1.4. The rate constant for rubidium release was the same in a sodium-containing as in a potassium-containing medium. The opposite effects of sodium and potassium ions on the poise of the equilibrium between the E1 and the E2 forms of the enzyme must, therefore, be due solely to opposite effects of these ions on the rate of conversion of E1 to E2.5. The rate constant for rubidium release was greatly increased by ATP and by ADP. Both nucleotides appeared to act at low-affinity sites and without phosphorylating the enzyme.6. Orthovanadate, in the presence of magnesium ions, stabilized the enzyme in the occluded-rubidium (E2Rb) form.7. Ouabain, in the presence of magnesium ions, prevented the occlusion of rubidium ions.8. We have measured the amount of rubidium occluded by the enzyme as a function of rubidium concentration, and estimate that at saturating rubidium concentrations about three rubidium ions can be occluded per phosphorylation site (or per ouabain-binding site).I. M. GL YNN AND D. E. RICHARDS 9. We have found that the occluded-rubidium form of the enzyme can also be formed by allowing rubidium ions to catalyze the hydrolysis of phosphoenzyme generated by the addition of ATP to enzyme suspended in a high-sodium medium.10. The properties of the occluded-rubidium form of the enzyme, and of the two routes that can lead to its formation, suggest that an analogous occluded-potassium form plays a central role in the transport of potassium ions through the sodiumpotassium pump. This hypothesis is supported by a detailed consideration of the probable magnitudes of the rate constants of the individual reactions making up the two routes.

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Section: Stoichiometrymentioning

confidence: 99%

Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Glynn

Richards

1982

The Journal of Physiology

155

113

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“…On thc one hand, 100 m M NaCl is much higher than the dissociation constant of the complex of sodium ions with ATPase (K,I = 0.2 mM 1301); on the other hand, it satisfies requirements of phosphorylation of protein from ATP [26] and of the formation of complex I with ouabain 1271. Finally, at potassium concentration of 20 mM the protein is entircly in the 'potassium' form, since k; = 0.05 mM [31].…”

Section: The -A Tpuse Preparationmentioning

confidence: 99%

Lack of Gross Protein Structure Changes in the Working Cycle of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase

Chetverin¹,

Venyaminov²,

Emelyanenko³

et al. 1980

European Journal of Biochemistry

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Infrared and tryptophan fluorescence spectra of practically all sufficiently stable functional con~plexes of a highly purified preparation of membrane-bound (Na', K +)-dependent ATPase have been measured. The formation of any functional complex was not accompanied by any considerable change of either shape or position of the tryptophan fluorescence spectrum. Only in the presence of adenine nucleotides was there a small decrease of fluorescence intensity (by 5 -8 %), which apparently results from a change of the sample light scattering. Analysis of the results obtained leads to the conclusion that the environment of no more than one or a few tryptophan residues may differ in all the (Na', K+)-ATPase complexes studies. A comparison of infrared protein spectra in the region of amide I band showed that at any wavenumber the differences between them did not exceed 3 % of the maximum absorption. This means that no more than 3 % of protein peptide groups can change their conformation upon transition between the enzyme functional states. These results, obtained by two independent techniques, allow us to conclude that even if changes of the internal protein structure occur during the working cycle of this transport system, they have an extremely local character.

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“…On the one hand, the stoichiometry of cation binding at these sites 13 Na' and 2 K+/molec u k (Na+,K')-ATPase [7,8]) coincides with the stoichiometry of transmembrane transport (3 Na ' and 2 K'/hydrolyzed ATP molecule [9]). On the other hand, binding of sodium and potassium only at the high-affinity sites affects particular reactions of the ATPase cycle [2].…”

Section: Resultsmentioning

confidence: 96%

“…Dissociation constants of ATPase complexes with Na' and K + , in the absence of any phosphorylating substrates, were determined from binding radioactive "Na' and 42K + with (Na',K+)-ATPase and were equal to 0.2 mM [7] and 0.05 mM [S] respectively. Thus, the concentrations of 2 mM NaCl and 0.5 mM KCl should be sufficient for almost all (Na', K+)-ATPase molecules to form complexes with the corresponding cations; however, these concentrations are evidently insufficient for cation binding at low-affinity sites, which are characterized by dissociation constants of the order of 10&30 mM [7,8].…”

Section: Resultsmentioning

confidence: 99%

Small Differences in Tryptophan Fluorescence Spectra of ‘Sodium’ and ‘Potassium’ Forms of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase

Chetverin¹,

Agalarov²,

Emelyanenko³

et al. 1980

European Journal of Biochemistry

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A detailed comparative analysis of tryptophan fluorescence spectra of 'sodium' and 'potassium' forms of (Na+, K+)-activated ATPase was carried out. The 'potassium' form spectrum is shifted relatice to that of the 'sodium' form by approximately 0.5 -1 nm towards shorter wavelengths. The maximal amplitude of the difference spectrum for these forms makes up about 2 % of maximal fluorescence intensity of any of the forms. The shape of the difference spectrum does not depend on the solution temperature or ionic strength. The spectral differences between the forms are reversible upon addition of a functionally opposite cation (K' for 'sodium' form and vice versa) into the medium, The results suggest that if the differences in fluorescence spectra of the 'sodium' and 'potassium' forms of (Na', K+)-ATPase resulted from the differences in the protein structure, they may be caused by an alteration in local environment of no more than one or two tryptophan residues.

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Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla

Cited by 41 publications

References 13 publications

Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Lack of Gross Protein Structure Changes in the Working Cycle of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase

Small Differences in Tryptophan Fluorescence Spectra of ‘Sodium’ and ‘Potassium’ Forms of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase

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Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla

Cited by 41 publications

References 13 publications

Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Occlusion of rubidium ions by the sodium‐potassium pump: its implications for the mechanism of potassium transport

Lack of Gross Protein Structure Changes in the Working Cycle of (Na+, K+)‐Dependent Adenosinetriphosphatase

Small Differences in Tryptophan Fluorescence Spectra of ‘Sodium’ and ‘Potassium’ Forms of (Na+, K+)‐Dependent Adenosinetriphosphatase

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Lack of Gross Protein Structure Changes in the Working Cycle of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase

Small Differences in Tryptophan Fluorescence Spectra of ‘Sodium’ and ‘Potassium’ Forms of (Na⁺, K⁺)‐Dependent Adenosinetriphosphatase