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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
    Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face (1991)
    Springer
    The journal of membrane biology 121 (1991), S. 163-176 
    Details
    ISSN: 1432-1424
    Keywords: Na,K-ATPase ; ion pumps ; electrogenic transport ; potentiometric dyes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na, K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na+ are electroneutral steps, and that release of Na+ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K+ indicate that binding and occlusion of K+ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na+ or K+ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel (“ion well”). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP+) or tetraphenylborate (TPB−), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP+ leads to a decrease of binding affinity for Na+ and K+, which is thought to result from the TPP−-induced change of electric field strength in the access channel.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870530
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  • 3
    Electronic Resource
    Electronic Resource
    Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements (1991)
    Springer
    The journal of membrane biology 121 (1991), S. 141-161 
    Details
    ISSN: 1432-1424
    Keywords: Na, K-ATPase ; ion pumps ; electrogenic transport ; voltage-sensitive dyes ; electrochromic effects
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01870529
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  • 4
    Electronic Resource
    Electronic Resource
    Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review (1985)
    Springer
    Cellular and molecular life sciences 41 (1985), S. 1-15 
    Details
    ISSN: 1420-9071
    Keywords: Rat carcinoma ; carcinoma, rat ; angiogenesis factors, tumor-derived ; Walker 256 carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02005853
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  • 5
    Electronic Resource
    Electronic Resource
    Geminate cations and anions in halocarbon systems: Life time, solvent effects and details of early stages (1986)
    Springer
    Journal of radioanalytical and nuclear chemistry 101 (1986), S. 329-338 
    Details
    ISSN: 1588-2780
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract Studies on the ionization processes in halocarbon systems, particularily with CCl4, have led to controversial interpretations in the past. Some of the reasons for discrepancies in CCl4-systems are known today. In a general way, they are typical for most halocarbons, though possibly not always equally dominant. The following aspects are discussed in the paper: complex formation, solvent and caging effects, geminate ion fragmentation, cross geminate charge transfer and delayed geminate ion neutralization through ion pair formation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02042434
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  • 6
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    Geminate cations and anions in halocarbon systems: Life time, solvent effects and details of early stages (1986)
    Springer
    Details
    Publication Date: 1986-10-01
    Print ISSN: 0236-5731
    Electronic ISSN: 1588-2780
    Topics: Chemistry and Pharmacology , Energy, Environment Protection, Nuclear Power Engineering
    Published by Springer
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  • 7
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    Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review (1985)
    Springer
    Details
    Publication Date: 1985-01-01
    Print ISSN: 0014-4754
    Topics: Biology , Medicine
    Published by Springer
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