Properties of the cytoplasmic ion binding sites.
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Binding Sites↗
Biomedical subjects
H J Apell
Publications and source records attributed to H J Apell.
Effects of free radicals on partial reactions of the Na,K-ATPase.
The function of the Na,K-ATPase is known to be considerably impaired in the presence of free radicals such as OH.. While previous experiments were largely based on the loss of enzymatic activity of the protein, this is the first communication dealing with partial reactions of the pump cycle in the presence of free radicals produced by water radiolysis. Three different system states, which are directly involved in ion transfer catalyzed by the enzyme, showed similar sensitivity to free radical action. This is indicated by largely identical D37-doses of the decay of the reaction amplitudes investigated. The decrease in the efficiency of the enzyme functions was largely due to a lethal damage of pump molecules. A kinetic analysis of the ATP-induced conformational transition E1-->E2 revealed, however, that a minor component of the inactivation is due to a reduction of the transition rate constant. The decrease of the enzymatic activity could be simulated by the decay of the rate-limiting conformational transition. This finding indicates the conservation of a close coupling between ATP-hydrolysis and sodium translocation process throughout free-radical induced inactivation. As a result of the tight coupling, enzyme modification at different system states leads to similar functional consequences for the protein.
Adenosine Triphosphate↗
Kinetics of the phosphorylation of Na,K-ATPase by inorganic phosphate detected by a fluorescence method.
Phosphorylation by Pi of the Na,K-ATPase from rabbit kidney in the absence of Na+ ions but in the presence of Mg2+ ions has been studied. In the absence of K+ ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the two states were monitored. Transient kinetic studies of phosphorylation were initiated by manual addition of Pi or by photochemical release of Pi from 1-(2-nitrophenyl)ethyl phosphate (caged Pi) using laser flash photolysis at 308 nm. Equilibrium studies of phosphorylation showed that the apparent Km for Pi was 23.0 +/- 0.3 microM (mean +/- sem) at pH 7.1 and 21 degrees C. The dye fluorescence increased in a biphasic manner on addition of 500 microM Pi to the enzyme: a rapid phase (t 1/2 < 1 s) and a slower exponential phase at 0.059 +/- 0.003 s-1. The rate of the rapid phase was studied by fast concentration-jump experiments and exhibited first-order kinetics in Pi up to 60 microM. Fluorescence records vs time were exponential, and a plot of the rate constant versus [Pi] had a slope of 1.47 x 10(5) M-1 s-1 and ordinate [Pi] = 0) intercept of 3.1 s-1. Addition of 50 mM NaCl to the phosphorylated enzyme induced an exponential decay in the dye fluorescence from which a rate constant of 0.10 +/- 0.005 s-1 was determined. These data were interpreted in terms of transformations between conformational states E1 and E2, and the phosphorylated state P-E2 defined in the Post-Albers mechanism of the Na,K-ATPase [Läuger, P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA] as follows: [formula: see text] The RH421 fluorescence of state P-E2 was studied over the pH range 6-8.5. Fluorescence was greatest at pH 8.5 and lowest at pH 6.0 in a simple binding isotherm with pK 7.5. The apparent Km for Pi rose cooperatively with increasing pH (pKa 8.6 and a Hill coefficient of 2). Therefore in the absence of monovalent metal ions, occupation of the cation (K+) binding sites by protons promotes phosphorylation by Pi.
Adenosine Triphosphate↗
Time-resolved fluorescence investigations of the interaction of the voltage-sensitive probe RH421 with lipid membranes and proteins.
Fluorescence lifetimes and fluorescence anisotropy decays of the voltage-sensitive styryl-pyridinium dye RH421 have been measured in the presence of dimyristoylphosphatidylcholine vesicles, the water soluble enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), and Na+,K(+)-ATPase-containing membrane fragments. The effect of an intramembrane electric field on the photophysical properties of the dye was investigated by the binding of the hydrophobic ion tetraphenylborate (TPB) to the membrane. TPB was found to significantly increase the average fluorescence lifetime and the order of membrane-bound dye. The increase in fluorescence lifetime is consistent with reorientation of the dye further into the membrane interior. The increase in order may be attributed to an electric field-induced alignment of the dye molecules. From the values of the rotational diffusion constant experimentally determined, the expected response time of the dye to an applied electric field can be calculated for a reorientational mechanism to be on the order of tens of nanoseconds. Experiments with rubisco showed that the dye interacts strongly with the protein. In this case, the dye is so tightly bound that it has almost no independent motion and rotates virtually solely with the protein. The rate of rotational motion of the dye in the presence of Na+,K(+)-ATPase-containing membrane fragments is similar to that in pure lipid membranes. The order parameter of the dye in the Na+,K(+)-ATPase membrane fragments is close to the maximum value. This is most probably due to the high density of protein molecules, which restricts the range of motion of the dye.
Binding Sites↗
Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump.
A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E1 of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (+/- standard deviation) for each step in the complete binding and release sequence: 0.12 +/- 0.01 mM (E2(K2)<==>KE1), 0.08 +/- 0.01 mM (KE1<==>E1A), 3.0 +/- 0.2 mM (NaE1<==>E1), 5.2 +/- 0.4 mM (Na2E1<==>NaE1) and 6.5 +/- 0.4 mM (Na3E1<==>Na2E1) at pH 7.2 and T = 16 degrees C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K+ than for Na+ ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na+ from the cytoplasm, and for import of K+ from the extracellular medium seems to be facilitated not by favorable binding affinities in state E1 but by the two ATP-driven reaction steps of the cycle, E2(K2) + ATP-->K2E1.ATP and Na3E1.ATP<==>(Na3) E1-P, which border the ion exchange reactions at the binding sites in conformation E1.
Animals↗
Sequential potassium binding at the extracellular side of the Na,K-pump.
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 degrees 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.
Animals↗
Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.
A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P <--> P-E2(Na3) <--> P-E2(Na2) <--> P-E2(Na) <--> P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) <--> P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.
Animals↗
Separation and characterization of Na+,K(+)-ATPase containing vesicles.
Na+,K(+)-ATPase was reconstituted in vesicles prepared by a dialysis method. Ion-exchange chromatography was used to obtain well characterized fractions from the inhomogeneous vesicle preparation. Lipid and protein content was determined by optical methods during the elution process. It was possible to separate fractions with distinct enzymatic and transport activities. A protocol was set up, which allowed to calculate the average number of 5-IAF labeled ion pumps per vesicle in the different fractions. The dependence of the number of protein molecules per vesicle was studied as function of the initial protein concentration added to the lipid solution before dialysis. The transport activity disappears completely at very low protein concentrations (3.3 micrograms protein per mg lipid). This observation is in favor of the proposal discussed in the literature, that the heterodimer (alpha beta)2 is the transport-active form of the Na+,K(+)-ATPase. The presented method can be applied to all reconstituted vesicle preparations in which the proteins can be labeled quantitatively with a fluorescence dye.
Chromatography, Ion Exchange↗
Inactivation of the Na, K-ATPase by radiation-induced free radicals. Evidence for a radical-chain mechanism.
Free radicals produced by water radiolysis were used to study the inactivation of the enzymatic activity of the Na,K-ATPase. A decrease of the activity to virtually zero with a mono-exponential dependence on the radiation dose was observed. The inactivation process is initiated by hydroxyl radicals. This was shown by the effect of appropriate radical scavengers such as t-butanol, formate and vitamin C. In all cases a significant increase in the characteristic D37 dose of inactivation was observed. Inactivation was found to show a so-called inverse dose-rate effect, i.e., the sensitivity of the enzyme to radical attack is increased if the dose rate is reduced. The data were found to agree with the relationship 1/D37 approximately 1/D1/2, which is known to be a strong indicator of a radical chain mechanism. This means that the inactivation, after initiation by single radicals, is amplified by a subsequent chain mechanism.
Cell Membrane↗
Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements.
A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (P(i) + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, and inhibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and binding is prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-fold faster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes.
Animals↗
Partial reactions of the Na,K-ATPase: determination of rate constants.
Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Investigation of reconstitution of the Na, K-ATPase in lipid vesicles.
Vesicles containing Na,K-ATPase were prepared by a dialysis method in buffers with various concentrations of K+ and Na+ ions. Ion-exchange chromatography has been used to separate proteoliposomes into protein-depleted and protein-rich fractions. The pumping activity of reconstituted ion pumps has been determined in the different fractions of the vesicle preparation using voltage-dependent fluorescence dyes. This method allowed to characterise vesicle fractions by a quantity which is proportional to the average number of pumps per vesicle with an active (inside-out) orientation. It could be shown that both, the amount of enzymatic active protein and the orientation of Na,K-ATPase in the vesicle lipid bilayer, is partially controlled by the Na+ and K+ concentration in the buffer during vesicle formation. High Na+ concentrations preferentially maintain the E1 conformation of the enzyme, which is less stable against denaturation during the dialysis, but displays a higher percentage of inside-out orientation of the transport-active protein. High K+ concentrations maintain the E2 conformation of the enzyme, which is stable against denaturation during the dialysis, but leads to a random orientation of the pump during dialysis.
Animals↗
Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase.
Intramolecular dynamics in Na,K-ATPase molecules have been studied by ultraviolet fluorescence spectroscopic methods: determination of temperature-dependent shifts in steady-state spectra, site-selective red-edge effects and their temperature dependence, and time-resolved emission decay as a function of excitation and emission wavelengths. The combination of these methods allows the characterization of the dipolar-relaxational mobility in the environment of the tryptophan residues. Our results show that the mean dipolar-relaxational time is of the order of one nanosecond at room temperature. This is much faster than what is usually observed in globular proteins. The fast dynamics of the protein dipoles are rapid enough so that the dipoles are in dielectric equilibrium during the slower ion transfer processes; this may have important functional consequences.
Buffers↗
Fluorescence study on cardiac glycoside binding to the Na,K-pump. Ouabain binding is associated with movement of electrical charge.
Recently we have presented evidence that the fluorescence probe RH 421 can be used to detect binding and release of ions at the extracellular face of the pump since these processes are associated with translocation of electrical charge. Applying this method to experiments with cardiac glycosides we found that: (1) ouabain induced fluorescence changes of the electrochromic dye, RH 421, were caused by the change of charges bound to the enzyme; (2) independent of the sodium concentration, the final fluorescence amplitude indicated that approximately 2 Na+ ions were bound to the pump; (3) the sodium release to the extracellular side involved two distinct electrogenic steps; (4) the kinetics of inhibition depended on the Na(+)-concentration. Experiments with hydrophobic ions indicated that the kinetics of ouabain binding to the Na-ATPase is voltage dependent; and (5) the applied technique is a convenient tool to characterize binding of cardiac glycosides to the Na,K-pump.
Animals↗
Na,K-ATPase in crystalline form investigated by scanning force microscopy.
Na,K-ATPase has been isolated in purified membrane fragments from kidney tissue and crystallized by phospholipase treatment to obtain two-dimensional, membrane-bound protein crystals. Scanning force microscopy has been used to identify and analyze the topography of the membrane fragments. Specific patterns in accordance with electron microscopic images have been found. In biological material under physiological conditions the scanning force is a crucial parameter for the resulting image at high resolution.
Animals↗
Electrostatic coupling of ion pumps.
In this paper the electrostatic interactions between membrane-embedded ion-pumps and their consequences for the kinetics of pump-mediated transport processes have been examined. We show that the time course of an intrinsically monomolecular transport reaction can become distinctly nonexponential, if the reaction is associated with charge translocation and takes place in an aggregate of pump molecules. First we consider the electrostatic coupling of a single dimer of ion-pumps embedded in the membrane. Then we apply the treatment to the kinetic analysis of light-driven proton transport by bacteriorhodopsin which forms two-dimensional hexagonal lattices. Finally, for the case of nonordered molecules, we also consider a model in which the pumps are randomly distributed over the nodes of a lattice. Here the average distance is equal to that deduced experimentally and the elemental size of the lattice is the effective diameter of one single pump. This latter model is applied to an aggregate of membrane-embedded Na, K- and Ca-pumps. In all these cases the electrostatic potential considered is the exact solution calculated from the method of electrical images for a plane membrane of finite thickness immersed in an infinite aqueous solution environment. The distributions of charges (ions or charged binding sites) are considered homogeneous or discrete in the membrane and/or in the external solution. In the case of discrete distributions we compare the results from a mean field approximation and a stochastic simulation.
Electrophysiology↗
Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements.
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.
Adenosine Triphosphate↗
Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face.
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.
Adenosine Triphosphate↗