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H J Apell

Publications and source records attributed to H J Apell.

At least 55 records · Page 3Linked to original sources
PubMed1990

Electrogenic transport by the Enterococcus hirae ATPase.

A transport ATPase from Enterococcus hirae was reconstituted in lipid vesicles and its electrogenic action investigated with the fluorescent dye oxonol VI as membrane potential probe. Reconstitution in bacterial and in soybean phospholipid mixtures led to transport-active vesicle preparations. Inside-out oriented ATPase molecules were activated by the addition of ATP to the extravesicular medium, generating in all experiments an intravesicularly positive potential. The extravesicular pH strongly influenced the initial pumping rate and the duration of the pumping activity. At neutral pH, transient pumping activity was observed, lasting for 1-2 min, while at pH 5.6, pumping was continuous. The transport activity was not dependent on the ionic composition of the buffer on either side of the membrane. These findings can be interpreted as the action of a proton ATPase, regulated by the cytoplasmic proton concentration and electrogenically translocating protons from the cytoplasm to the extracellular space.

Adenosine Triphosphatases↗
PubMed1990

Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode.

Na,K-ATPase from rabbit kidney outer medulla was reconstituted in large unilamellar lipid vesicles by detergent dialysis. Vesicles prepared in the presence or absence of potassium allowed to study two different transport modes: the (physiological) Na,K-mode in buffers containing Na+ and K+ and the Na-only mode in buffers containing Na+ but no K+. The ATP hydrolysis activity was obtained by determination of the liberated inorganic phosphate, Pi, and the inward directed Na+ flux was measured by 22Na-tracer flux. Electrogenic transport properties were studied using the membrane potential sensitive fluorescence-dye oxonol VI. The ratio upsilon(Na,K)/upsilon(Na) of the turnover rates in the Na,K-mode and in the Na-only mode is 6.6 +/- 2.0 under otherwise identical conditions and nonlimiting Na+ concentrations. Strong evidence is found that the Na-only mode exhibits a stoichiometry of 3Na+cyt/2Na+ext/1ATP, i.e. the extracellular (= intravesicular) Na+ has a potassium-like effect. In the Na-only mode one high-affinity binding side for ATP (KM congruent to 50 nM) was found, in the Na,K-mode a high- and low-affinity binding side with equilibrium dissociation constants, KM, of 60 nM and 13 microM, respectively. The sensitivity against the noncompetitively inhibiting ADP (KI = 6 microM) is higher by a factor of 20 in the Na-only mode compared to the Na,K-mode. From the temperature dependence of the pumping activity in both transport modes, activation energies of 160 kJ/mol for the Na,K-mode and 110 kJ/mol for the Na-only mode were determined.

Animals↗
PubMed1989

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy.

Na+/K+-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the enzyme in a density of 10(3)-10(4) protein molecules per microm2 (Deguchi et al. (1977) J. Cell. Biol. 75, 619-634). In this paper we show that these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enzyme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-40% of a monolayer of membrane fragments, characteristic infrared bands of the protein such as the amide I and II bands can be resolved. About 24% of the NH-groups of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO- band at 1400 cm-1. Experiments with aqueous media of different ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris+ or Na+.

Animals↗
PubMed1989

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles.

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.

Animals↗
PubMed1989

Conformational transitions and change translocation by the Na,K pump: comparison of optical and electrical transients elicited by ATP-concentration jumps.

The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (approximately 8000 microm(-2)) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive electrode. ATP was released in the medium from a photolabile inactive ATP derivative ("caged" ATP) by a 40-microsec light flash. Electrical signals resulting from transient charge movements in the protein under single-turnover conditions were recorded in the external measuring circuit. In parallel experiments carried out under virtually identical conditions, the fluorescence of membrane fragments containing Na,K-ATPase with covalently-bound 5-iodoacetamido-fluorescein (5-IAF) was monitored after the ATP-concentration jump. When the medium contained Na+, but no K+, the fluorescence of the 5-IAF-labeled protein decreases monotonously after release of ATP. In the experiments with membrane fragments bound to a planar bilayer, a transient pump current was observed which exhibited virtually the same time behavior as the fluorescence decay. This indicates that optical and electrical transients are governed by the same rate-limiting reaction step. Experiments with chymotrypsin-modified Na,K-ATPase suggest that both the fluorescence change as well as the charge movement are associated with the deocclusion of Na+ and release to the extracellular side. In experiments with Na+-free K+ media, a large inverse fluorescence change is observed after the ATP-concentration jump, but no charge translocation can be detected. This indicates that deocclusion of K+ is an electrically silent process.

Animals↗
PubMed1989

Lateral order in mixed lipid bilayers and its influence on ion translocation by gramicidin: a model for the structure-function relationship in membranes.

The temperature-composition phase diagram of dimyristoylphosphatidylcholine and dipentadecylphosphatidylglycerol (DiC15PG) was determined by mass densitometry. For a mixture containing 30 mol% DiC15PG, the homogeneous distribution of the 2 components is demonstrated in the fluid state at T = 35 degrees C by small-angle neutron scattering in combination with the inverse contrast variation method. By the same technique, the coexistence of fluid and condensed phases at T = 23.3 degrees C could be shown in agreement with the densitometric data. Furthermore, it is demonstrated that Ca++ induces, even at T = 35 degrees C, separation into 2 fluid phases. A corresponding phase separation is found in bimolecular lipid membranes ("black films") by analysis of the single-channel conductance fluctuations of gramicidin A incorporated into an equimolarly mixed membrane of neutral lecithin and charged phosphatidic acid. The results are discussed as primary examples on the model-membrane level for the important structure-function relationship of biomembranes.

Calcium↗
PubMed1989

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles.

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.

Fluorescent Dyes↗
PubMed1988

Reconstitution of Na+/K+-ATPase into phosphatidylcholine vesicles by dialysis of nonionic alkyl maltoside detergents.

The reconstitution of Na+/K+-ATPase from outer medulla of rabbit kidney into large unilamellar liposomes was achieved through detergent removal by dialysis of mixed micellar solutions of synthetic dioleoyl phosphatidylcholine/octyl glucoside and Na+/K+-ATPase/decyl maltoside or decenyl maltoside. Tight, transport-active liposomes were formed when the lipid and the enzyme were solubilized separately in the nonionic detergents and mixed immediately before starting the dialysis. The two maltoside detergents with different structures of the hydrophobic part of the molecule proved to be well suited for the solubilization of Na+/K+-ATPase with high retention of enzyme activity; the inactivation of enzyme being evidently slower with the unsaturated decenyl maltoside. The diameters of the proteoliposomes, 110 and 170 nm, respectively, were also dependent on the structure of the maltoside detergent, the saturated decyl maltoside producing the bigger liposomes. After freeze-fracture, both preparations exhibited intramembranous particles as structural indicators of successful reconstitution. The electrogenic activity of the reconstituted enzyme was determined by fluorescence measurements with Oxonol VI and by tracer-flux measurements with 22Na+.

Animals↗
PubMed1988

Voltage dependence of partial reactions of the Na+/K+ pump: predictions from microscopic models.

A theoretical treatment of the voltage dependence of electroneutral Na+-Na+ and K+-K+ exchange mediated by the Na+/K+ pump is given. The analysis is based on the Post-Albers reaction scheme in which the overall transport process is described as a sequence of conformational transitions and ion-binding and ion-release steps. The voltage dependence of the exchange rate is determined by a set of 'dielectric coefficients' reflecting the magnitude of charge translocations associated with individual reaction steps. Charge movement may result from conformational changes of the transport protein and/or from migration of ions in an access channel connecting the binding sites with the aqueous medium. It is shown that valuable mechanistic information may be obtained by studying the voltage dependence of transport rates at different (saturating and nonsaturating) ion concentrations.

Electrophysiology↗
PubMed1988

Transient behaviour of the Na+/K+-pump: microscopic analysis of nonstationary ion-translocation.

In recent years fast perturbation techniques have been applied for investigating the mechanism of the Na+/K+-pump. Experiments in which nonstationary pump-currents and ion fluxes are measured after a voltage or ATP-concentration jump yield kinetic information which cannot be obtained from ordinary steady-state experiments. In this paper a theoretical treatment is described by which transient pump-currents and ion fluxes can be analyzed in a unified way. The method is based on the assumption that the operation of the pump involves a sequence of conformational transitions and ion-binding and -release steps. The charge displacements associated with the individual reaction steps are described by a set of dielectric coefficients. The nonstationary behaviour of the Na+/K+-pump is analyzed on the basis of the Albers-Post reaction cycle. It is shown that the different studies of transient pump-currents and ion fluxes carried out so far lead to internally consistent conclusions with respect to the nature of the electrogenic steps of the transport cycle.

Adenosine Triphosphate↗
PubMed1988

Current transients generated by the Na+/K+-ATPase after an ATP concentration jump: dependence on sodium and ATP concentration.

Planar membrane fragments containing a high density of oriented Na+/K+-ATPase molecules are bound to planar lipid bilayers. ATP is released in the aqueous solution within milliseconds from an inactive, photolabile precursor ('caged ATP') by an intense light flash. By this ATP-concentration jump a large number of pump molecules is activated almost simultaneously. Charge translocation in the pump molecule results in a voltage transient which is recorded in the external measuring circuit. From the voltage signal, the intrinsic pump current Ip(t) can be evaluated using information on the circuit parameters of the compound membrane system. The pump current Ip(t) is compared with the results of numerical simulations of a reaction cycle derived from the Post-Albers reaction scheme combined with the photochemical release reaction of caged ATP. The time course of Ip can be satisfactorily fitted using kinetic parameters of the Na+/K+-ATPase from the literature. The dependence of Ip on sodium concentration cNa can be described using a single set of kinetic parameters in which only cNa is varied. Ip as a function of cNa is well fitted by a first-order Michaelis-Menten type equation with Km approximately equal to 4 mM. This finding is consistent with the assumption that two sodium binding sites have a high affinity and that a third site of lower affinity is rate limiting. The ATP concentration dependence of Ip is studied by varying the concentration of caged ATP in the solution and the yield of photochemical release of ATP.

Adenosine Triphosphate↗
PubMed1987

Oxonol VI as an optical indicator for membrane potentials in lipid vesicles.

Experiments with large unilamellar dioleoylphosphatidylcholine vesicles were carried out in order to study the effect of membrane potential on the fluorescence of Oxonol VI. A partition equilibrium of dye between membrane and water was found to exist with a partition coefficient gamma identical to c lipid/c water of about 19,000 (at zero voltage). In the presence of an inside-positive membrane potential, the negatively charged dye accumulates in the intravesicular aqueous space according to a Nernst equilibrium. This leads to an increased adsorption of dye to the inner lipid monolayer and to a concomitant increase of fluorescence. The fluorescence change can be calibrated as a function of transmembrane voltage by generating a potassium diffusion potential in the presence of valinomycin. The intrinsic fluorescence of the membrane-bound dye is not affected by voltage; the whole influence of voltage on the fluorescence results from voltage-dependent partitioning of the dye between water and membrane. The voltage dependence of the apparent partition coefficient can be quantitatively described by a three-capacitor model in which the dye is assumed to bind to adsorption planes located on the hydrocarbon side of the membrane/solution interface. Oxonol VI was found to be suitable for detecting changes of membrane potential associated with the activity of the (Na+ + K+)-ATPase in reconstituted vesicles. When ATP is added to the external medium, pump molecules with the ATP-binding side facing outward become activated; this results in a translocation of net positive charge towards the vesicle interior. Under this condition, fluorescence changes corresponding to (inside-positive) potentials of up to 150-200 mV are observed. After the build-up of the membrane potential, a quasi-stationary state is reached in which the pump current is compensated by a back-flow of charge through passive conductance pathways.

Adsorption↗
PubMed1987

Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP.

Nonstationary electric currents are described which are generated by the Na,K-pump. Flat membrane sheets 0.2-1 micron in diameter containing a high density of oriented Na,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 current Ip(t) can be evaluated from the recorded current signal, using estimated values of the circuit parameters of the compound membrane system. Ip(t) exhibits a biphasic behavior with a fast rising period, followed by a slower decline towards a small quasi-stationary current. The time constant of the rising phase of Ip(t) is found to depend on the rate of photochemical ATP release. Further information on the microscopic origin 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.

Adenosine Triphosphate↗
PubMed1987

Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents.

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.

Animals↗