Na,K-ATPase on a waveguide sensor : supramolecular assembly and side directed binding studies by surface-confined fluorescence

Grell E, Pawlak M, Anselmetti D, Schick E, Lewitzki E, Ehrat M. Na,K-ATPase on a waveguide sensor : supramolecular assembly and side directed binding studies by surface-confined fluorescence. In: Taniguchi K, Kaya S, eds. Na/K-ATPase and related ATPases: proceedings of the 9th International Conference on the Na/K-ATPase and Related ATPases. Excerpta Medica international congress series. Vol 1207. Amsterdam: Elsevier; 2000: 437-440.The functional assembly of FITC-Na,K-ATPase membrane fragments on a surface-modified Ta2O5 waveguide allows to investigate the directed binding of ligands by surface-confined fluorescence studies. The results allow to draw conclusions about the sidedness of interactions. The fluorescence intensity decrease observed upon the selective binding of K+ is attributed to its coordination to a site accessible from the former intracellular membrane side

The Influence of Manga on the Graphic Novel

This material has been published in The Cambridge History of the Graphic Novel edited by Jan Baetens, Hugo Frey, Stephen E. Tabachnick. This version is free to view and download for personal use only. Not for re-distribution, re-sale or use in derivative works. © Cambridge University PressProviding a range of cogent examples, this chapter describes the influences of the Manga genre of comics strip on the Graphic Novel genre, over the last 35 years, considering the functions of domestication, foreignisation and transmedia on readers, markets and forms

Molecular Ionics of Anion Receptor Molecules. A microcalorimetric study.

The coordination of divalent and monovalent inorganic anions to synthetic polyammonium receptors is investigated in aqueous solution around neutral pH by titration calorimetry and NMR spectroscopy. High-affinity 1:1 complexes are formed by a pyrrole type cryptand (1) with sulfate and phosphate, characterized by association constants of almost 107 M-1. Affinities close to 105 M-1 are found for polyazacryptands (3 and 4) exhibiting F-/Cl- selectivity. The binding affinities and the anion selectivities are mainly caused by the charges of ligands and anions, which is discussed on the basis of simple calculations of the electrostatic contribution to the anion/receptor interactions. The binding of all investigated anions is exothermic at 298.2 K. The contribution of the large negative ΔH values to the free energy of anion binding of the pyrrole type ligand is partially compensated by marked negative ΔS values. These unfavorable entropic contributions are attributed to the additional inclusion of water molecules in the anion/receptor complexes

Rates and Equilibrium of Cu(A) to Heme a Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Intramolecular electron transfer between Cu(A) and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. Cu(A), the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the Cu(A) center and the concomitant reduction of heme a. The rate constants for heme a reduction and Cu(A) reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between Cu(A) and heme a. The rate constants for Cu(A) → heme a ET and the reverse heme a → Cu(A) process were found to be 20,400 s(−1) and 10,030 s(−1), respectively, at 25°C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies

Inhibition and partial reactions of Na,K-ATPase studied by FTIR difference spectroscopy

Reaction‐induced infrared (IR) difference spectroscopy with caged ATP and Na,K‐ATPase allows us to differentiate unambiguously between phosphorylated and unphosphorylated states of the enzyme as well as of its ouabain complex. The IR spectral changes upon phosphoenzyme formation are characterized and interpreted. Our results show clearly that high Na+ concentrations prevent the binding of ouabain with high affinity, which is consistent with the results of a corresponding kinetic study employing spectrofluorimetry and calorimetric titrations. This unexpected antagonism leading to low ouabain affinity is assumed related to a conformation of the protein, induced by low affinity binding of the third Na+ ion. We thus conclude that not the free enzyme but a phosphorylated state of the reaction cycle preferentially binds ouabain and leads to the loss of hydrolytic activity

Structural Changes in the Catalytic Cycle of the Na+,K+-ATPase Studied by Infrared Spectroscopy

AbstractPig kidney Na+,K+-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na3+ to an E2P state was initiated by photolysis of P3-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg2+ and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na+,K+-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15°C. Several controls ensured that the observed difference signals were due to structural changes of the Na+,K+-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca2+-ATPase and H+,K+-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm−1, respectively, for αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na3+ to an E2P state. A negative band at 1730 cm−1 is in line with the presence of a protonated Asp or Glu residue that coordinates Na+ in E1Na3+. Infrared signals were also detected in the absorption regions of ionized carboxyl groups