Nucleotide/Protein Interaction: Energetic and Structural Features of Na,K-ATPase

Microcalorimetric titrations allow to recognize and investigate high-affinity ligand binding to Na,K-ATPase. Titrations with the cardiac glycoside Ouabain, which acts as a specific inhibitor of the enzyme, have provided not only the thermodynamic parameters of high-affinity binding with a stoichiometric coefficient of about 0.6 but also evidence for low-affinity binding to the lipid. The marked enthalpic contribution of -95 kJ mol^-1 at 298.2 K is partially compensated by a large negative entropy change, attributed to an increased interaction between water and the protein. The calorimetric ADP and ATP titrations at 298.2 K are indicative of high-affinity nucleotide binding either in 3 mM NaCl, 3 mM MgCl₂ or at high ionic strength such as 120 mM choline chloride. However, no binding is detected in the buffer solution alone at low ionic strength. The affinities for ADP and ATP are similar, around 10⁶ M^-1 and the stoichiometric coefficients are close to that of Ouabain binding. The exothermic binding of ADP is characterized by a ΔH and ΔS value of -65 kJ mol^-1 and -100 J mol^-1 K^-1, respectively. TheΔH value for ATP binding is larger than for ADP and is compensated by a larger, unfavorable ΔS value. This leads to an enthalpy/entropy compensation, which could express that H-bond formation represents the major type of interaction. As for Ouabain, the negative ΔS values that are also characteristic of nucleotide binding can indicate an increase of solvate interaction with the protein due to a conformational transition occurring subsequent to the binding process. The resulting binding constants are discussed with regard to the results of other studies employing different techniques. A molecular interaction model for nucleotide binding is suggested. This revised version was published online in July 2006 with corrections to the Cover Date.     

lin-Benzo-ATP and-ADP: Versatile fluorescent probes for spectroscopic and biochemical studies

lin-Benzo-adenine nucleotides can act not only as probes for fluorescence studies but also as structural active site probes for enzymes. To understand the basic properties oflin-benzo-ATP and-ADP, protolysis and Mg²⁺ and Ca²⁺, binding are investigated between pH 6.2 and pH 8.5 by spectrophotometric and spectrofluorometric titrations. Based on a reaction model, a set of equilibrium constants is determined which is consistent with all available experimental results. The pK values of the Mg²⁺ and Ca²⁺ complex oflin-benzo-ATP in the chosen medium are 4.6 and 4.1, respectively, and those for the corresponding diphosphate are 3.1 and 2.8, respectively. Fluorescence and absorption spectra are reported.This is a peer-reviewed conference proceeding article from the Third Conference on Methods and Applications of Fluorescence Spectroscopy, Prague, Czech Republic, October 18–21, 1993.     

Removing coordinated metal ions from proteins: a fast and mild method in aqueous solution

Thermodynamic and kinetic studies of metal binding to proteins require the investigation of metal-free proteins, which are often difficult to obtain. We have developed a very fast and mild method to eliminate metal ions from proteins by column chromatography using a commercially available Ni-NTA-type stationary phase. This material, initially designed for protein purification purposes in biotechnology, acts as a strong cation chelator when Ni²⁺ ions are removed. We have tested this new method with Ca-ATPase, an integral membrane protein exhibiting a strong affinity for Ca²⁺. By eluting the protein over the Ni²⁺-free NTA gel, we could remove 95% of the total Ca²⁺ and obtain an essentially Ca²⁺-free protein. This method is efficient with only a small amount of NTA gel, and we suggest that it can be applied in general for removal of metal ions from proteins. Moreover, as this procedure can be carried out under mild conditions, the chosen protein kept its enzymatic activity.     

Characterizing Protein Conformational Transitions of Na,K-ATPase with Antibodies by Fluorescence Spectroscopy

Stationary and time-resolved fluorescence of FITC–Na,K-ATPase is investigated as a function of pH in the presence of different ligands, cations, and the monoclonal anti-FITC antibody 4-4-20. The binding of K⁺ and of the antibody leads to the same decreased fluorescence intensity level. Antibody binding is observed only under conditions where the enzyme exists in the conformational state F₁, and not in the form of the Na⁺ or K⁺ complex or when it is phosphorylated with inorganic phosphate in the presence of Mg²⁺. For the interpretation of the results it is shown that the fluorophore is not essentially affected by an acidity change of the bound dye, so that pK variations responsible for the observed intensity changes can be excluded in favor of a static quenching process     

DEDO: A specific,fluorescent inhibitor for spectroscopic investigations of Na,K-ATPase

The interaction between the fluorescent ouabain derivative DEDO and purified renal Na,K-ATPase (isolated from different animal species) is investigated. Equilibrium binding studies provide a pK value of about 7.5 and a stoichoimetric coefficient of 1. Nonmodified ouabain exhibits the same affinity to the rabbit enzyme; the enzyme originating from the other sources binds DEDO 10 times less strongly than ouabain. Kinetic studies indicate that this is the consequence of a 10 times higher dissociation rate constant of the complexes formed with DEDO. The fluorescence emission intensity of DEDO is enhanced, being dependent on the enzyme source. The single decay time of DEDO is 3 ns in the absence and 21 ns in the presence of the rabbit enzyme and 14 ns in the presence of the pig renal enzyme. This result suggests that the fluorophore of DEDO is bound to a very hydrophobic environment of the enzyme. Further characterization of the static fluorescence spectra provides evidence for energy transfer between Trp residues of the enzyme and DEDO. Distance estimations suggest that one or two Trp residues are likely to be located in the proximity of the fluorophore.     

Lipid-similar Thermal Transition of Polyethylene Glycol Alkyl Ether Detergents

A narrow, reversible endothermic main transition is found in the aqueous micellar phase of octaethylene glycol tetradecyl ether (C₁₄E₈) by DSC, characterized by a transition temperature of 41°C and a H value of 0.5 kcal mol^–1, which is not observed by light scattering. This transition is assigned to a cooperative conformational rearrangement of the assembled amphiphilic detergent molecules and not to a micelle aggregation process. It is suggested that the detergents polar head group is primarily involved in this rearrangement.This revised version was published online in November 2005 with corrections to the Cover Date.     

Toward an understanding of the fluorescence intensity changes observed on fluorescein 5′-Isothiocyanate-Na+,K+-ATPase

The fluorescence emission intensity between the Na⁺, and the K⁺ complex of Na⁺,K⁺-ATPase, labeled with fluorescein 5-isothiocyanate (FITC), differs by 30 to 40%. Experimental studies are carried out to elucidate the physical reasons which account this intensity difference. The dissociation constant of protolysis of the covalently bound FITC and its fluorescence decay times are determined in media of different ionic compositions and are compared with the corresponding properties of a synthetic model compound. The fluorophore bound to the protein is characterized by two decay times in the nanosecond range; the model compound, by a single one. The static fluorescence intensity changes are discussed on the basis of these results.This is a peer-reviewed conference proceeding article from the Third Conference on Methods and Applications of Fluorescence Spectroscopy, Prague, Czech Republic, October 18–21, 1993.     

Phase Transitions in Non-ionic Detergent Micelles

Differential scanning calorimetry (DSC) studies of micellar, 60 mM solutions of the octaethyleneglycol alkylethers C₁₄E₈ and C₁₆E₈ provide evidence for a narrow endothermic transition at 41 and 32°C,respectively, characterized by an enthalpy change of 2 kJ mol⁻¹ for both detergents. The observed thermal transition is indicative of a concerted transition of the surfactant molecules, as illustrated on the basis of a simple molecular model. The effect of co-solvents such as different alcohols on the thermal transition is investigated. Glycerol markedly lowers the transition temperature whereas the transition is absent in the presence of at least 10% ethanol. The calorimetric transition correlates with the temperature dependent increase of viscosity and static light scattering as well as with changes observed by small-angle neutron scattering (SANS). The SANS results provide clear evidence for a distinct structural change occurring at the transition temperature, which is interpreted as a sphere-to-rod transition of the detergent micelles. Moreover, the rod length increases with increasing temperature. We suggest that the process causing the thermal transition acts as the prerequisite of the growth process. This revised version was published online in August 2006 with corrections to the Cover Date.     

Conformational changes of Na,K-ATPase probed with eosin Y

Time-resolved fluorescence and binding studies have been carried out on Na,K-ATPase in the presence of the fluorescent dye eosin Y to obtain thermodynamic and kinetic parameters for the interaction of the enzyme with different cations. Eosin Y binding is indicated by a 3 ns fluorescence decay process and is observed only in the presence of mono- and divalent cations. This type of cation binding is interpreted as a nonselective electrostatic interaction, with negatively charged groups of the enzyme providing a high-affinity eosin Y binding site. Eosin Y binding is observed only under conditions where the enzyme exists in the conformational state F₁. The kinetic parameters of eosin Y binding have been determined employing stopped-flow fluorometry.