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  • 1
    Electronic Resource
    Electronic Resource
    Sequential potassium binding at the extracellular side of the Na,K-Pump (1995)
    Springer
    The journal of membrane biology 145 (1995), S. 165-173 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; Potassium binding ; Electrogenic transport ; Cation binding site ; Sequential binding ; Activation energy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Ion binding at the extracellular face of the Na,K-ATPase is electrogenic and can be monitored by the styryl dye RH 421 in membrane fragments containing a high density of the Na,K-pumps. The fluorescent probe is noncovalently bound to the membrane and responds to changes of the local electric field generated by binding or release of cations inside the protein. Due to the fact that K+ binding from the extracellular side is an electrogenic reaction, it is possible to detect the amount of ions bound to the pump as function of the aqueous concentration. The results are in contradiction to a second order reaction, i.e., a simultaneous binding of two K+ ions. A mathematical model is presented to discuss the nature of the two step binding process. On the basis of this model the data allow a quantitative distinction between binding of the first and the second K+ ion. The temperature dependence of ion binding has been investigated. At low temperatures the apparent dissociation constants differ significantly. In the temperature range above 20°C the resulting apparent dissociation constants for both K+ ions merge and have values between 0.2 and 0.3 mm, which is consistent with previous experiments. The activation energy for the half saturating concentration of K+ is 22 kJ/mol. Additional analysis of the titration curve of K+ binding to the state P — E2 by the Hill equation yields a Hill coefficient, nHill, of 1.33, which is in agreement with previously published data.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00237374
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  • 2
    Electronic Resource
    Electronic Resource
    Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement (1999)
    Springer
    The journal of membrane biology 168 (1999), S. 221-228 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Na,K-ATPase — Cytoplasmic ion binding — Electrochromic fluorescent dye — FITC — Ion transport — Energy transduction mechanism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na+-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na+ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na+ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na+ ions and followed by the conformational transition E1/E2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na+ ions are bound.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900511
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  • 3
    Electronic Resource
    Electronic Resource
    Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase (1985)
    Springer
    The journal of membrane biology 85 (1985), S. 49-63 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; reconstitution ; potential sensitive dye ; ion fluxes ; transport kinetics ; activation energy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01872005
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  • 4
    Electronic Resource
    Electronic Resource
    Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP (1987)
    Springer
    The journal of membrane biology 97 (1987), S. 161-178 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; ion pumps ; electrogenic transport ; concentration jump ; “caged” ATP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Nonstationary electric currents are described which are generated by the Na,K-pump. Flat membrane sheets 0.2–1 μm in diameter containing a high density of oriented N,K-ATPase molecules are bound to a planar lipid bilayer acting as a capacitive electrode. In the aqueous phase adjacent to the bound membrane sheets, ATP is released within milliseconds from an inactive, photolabile precursor (“caged” ATP) by an intense flash of light. After the ATP-concentration jump, transient current and voltage signals can be recorded in the external circuit corresponding to a translocation of positive charge across the pump protein from the cytoplasmic to the extracellular side. These electrical signals which can be suppressed by inhibitors of the Na,K-ATPase require the presence of Na+ but not of K+ in the aqueous medium. The intrinsic pump currentI p (t) can be evaluated from the recorded current signal, using estimated values of the circuit parameters of the compound membrane system.I p (t) exhibits a biphasic behavior with a fast rising period, followed by a slower decline towards a small quasistationary current. The time constant of the rising phase ofI p (t) is found to depend on the rate of photochemical ATP release. Further information on the microscopic orgin of the current transient can be obtained by double-flash experiments and by chymotrypsin modification of the protein. These and other experiments indicate that the observed charge-translocation is associated with early events in the normal transport cycle. After activation by ATP, the pump goes through the first steps of the cycle and then enters a long-lived state from which return to the initial state is slow.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869220
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  • 5
    Electronic Resource
    Electronic Resource
    Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents (1987)
    Springer
    The journal of membrane biology 97 (1987), S. 179-191 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; ion pumps ; electrogenic transport ; Albers-Post cycle ; partial reactions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Nonstationary pump currents which have been observed in K+-free Na+ media after activation of the Na,K-ATPase by an ATP-concentration jump (see the preceding paper) are analyzed on the basis of microscopic reaction models. It is shown that the behavior of the current signal at short times is governed by electrically silent reactions preceding phosphorylation of the protein; accordingly, the main information on charge-translocating processes is contained in the declining phase of the pump current. The experimental results support the Albers-Post reaction scheme of the Na,K-pump, in which the translocation of Na+ precedes translocation of K+. The transient pump current is represented as the sum of contributions of the individual transitions in the reaction cycle. Each term in the sum is the product of a net transition rate times a “dielectric coefficient” describing the amount of charge translocated in a given reaction step. Charge translocation may result from the motion of ion-binding sites in the course of conformational changes, as well as from movement of ions in access channels connecting the binding sites to the aqueous media. A likely interpretation of the observed nonstationary currents consists in the assumption that the principal electrogenic step is the E1-P/P-E2 conformational transition of the protein, followed by a release of Na+ to the extracellular side. This conclusion is supported by kinetic data from the literature, as well as on the finding that chymotrypsin treatment which is known to block the E1-P/P-E2 transition abolishes the current transient. By numerical simulation of the Albers-Post reaction cycle, the proposed mechanism of charge translocation has been shown to reproduce the experimentally observed time behavior of pump currents.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869221
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  • 6
    Electronic Resource
    Electronic Resource
    Electrogenic properties of the Na,K pump (1989)
    Springer
    The journal of membrane biology 110 (1989), S. 103-114 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; electrogenic ion pumps ; voltage dependence ; ion flux ; pump current
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869466
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  • 7
    Electronic Resource
    Electronic Resource
    Dicarboxylic acid analogs of Gramicidin A: Dimerization kinetics and single channel properties (1979)
    Springer
    The journal of membrane biology 50 (1979), S. 271-285 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary According to the model of Urry, the cation-permeable gramicidin channel is a dimeric helix formed by association of two peptide monomers linked at their amino ends. In this paper the channel properties of gramicidin analogs are described which have been obtained by chemical modification at the coupling site of the two half-channels. In these analogs the amino terminal-CHO group is replaced by-CO(CH2) n COOH(n=2, 3, 4, 5, 6). All analogs form conducting channels in black lipid membranes with the same general properties as found for gramicidin A. The observation that the channel-forming activity decreases with increasing pH is consistent with the notion that the half-channels are linked at the amino terminus. The channel lifetime of the different analogs varies between 2 msec and ≧50 sec, the longest lifetime being found for the compound withn=3. The single-channel conductance Λ is always smaller than that of gramicidin A, but the reduction of Λ depends on the nature of the permeable ion. Ion specificity was studied at 1m electrolyte by measuring the conductance Λ for different permeable ions (Na+, K+, Cs+). The conductance ratioΛ(Cs+)/Λ(Na+) was found to vary between 2 and 10.5 for the different analogs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01868893
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  • 8
    Electronic Resource
    Electronic Resource
    Erratum (1985)
    Springer
    The journal of membrane biology 86 (1985), S. 189-189 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870784
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  • 9
    Electronic Resource
    Electronic Resource
    Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method (1999)
    Springer
    The journal of membrane biology 168 (1999), S. 169-181 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Calcium binding — Ion transport — Binding site — Electrogenicity — Styryl dyes — pH dependence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: 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 Ca2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca2+ ions in state E1 with an apparent half-saturation concentration, K M of 600 nm. In state P-E2 two K M values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E2, it could be demonstrated that H+ binding is electrogenic and that Ca2+ 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 E1 with unoccupied ion sites, independent of the buffer composition.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900507
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  • 10
    Electronic Resource
    Electronic Resource
    Formation of ion channels by a negatively charged analog of gramicidin a (1977)
    Springer
    The journal of membrane biology 31 (1977), S. 171-188 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary O-pyromellitylgramicidin is a derivative of gramicidin in which three carboxyl groups are introduced at the terminal hydroxyl end of the peptide. Experiments with artificial lipid membranes indicate that this negatively charged analog forms ion-permeable channels in a way similar to that of gramicidin. If O-pyromellitylgramicidin is added to only one aqueous solution, the membrane conductance remains small, but increases by several orders of magnitude if the same amount is also added to the other side. In accordance with the dimer model of the channel, the membrane conductance under symmetrical conditions is proportional to the square of the aqueous concentration of O-pyromellitylgramicidin over a wide range. The ratioΜ PG/Μ G of the single-channel conductance of O-pyromellitylgramicidin to that of gramicidin is close to unity at high ionic strength, but increases more than fivefold at smaller ionic strength (0.01m). This observation is explained in terms of an electrostatic effect of the fixed negative charges localized near the mouth of the channel. In a mixture of O-pyromellitylgramicidin and gramicidin, unit conductance steps of intermediate size are observed in addition to the conductance steps corresponding to the pure compounds, indicating the formation of hybrid channels. Hybrid channels with preferred orientation may be formed if small amounts of gramicidin and O-pyromellitylgramicidin are added to opposite sides of the membrane. These hybrid channels show a distinct asymmetry in the current-voltage characteristic.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869403
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