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P Gräber

Publications and source records attributed to P Gräber.

At least 19 recordsLinked to original sources
PubMed1992

Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H(+)-ATPase from chloroplasts.

The H(+)-ATPase from chloroplasts CFoF1, was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni-site ATP synthesis was initiated by the addition of 20 nM [alpha-32P]ADP, and enzyme-bound and free nucleotides were separated by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.55 +/- 0.05. In a second experiment, uni-site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [alpha-32P]ATP; enzyme-bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme-bound ADP to ATP was 0.46 +/- 0.04. In a third experiment, uni-site ATP hydrolysis under de-energized conditions was initiated by the addition of 39 nM [alpha-32P]ATP and NH4Cl/valinomycin in the absence of illumination. Free and enzyme-bound nucleotides were separated also by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.43 +/- 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.

Adenosine Triphosphate↗
PubMed1992

Kinetics of ATP synthesis catalyzed by the H(+)-ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin.

The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).

Adenosine Triphosphate↗
PubMed1992

Activation of the H(+)-ATP synthase in the photosynthetic bacterium Rhodobacter capsulatus.

The regulation of the membrane-bound H(+)-ATPase from the photosynthetic bacterium Rhodobacter capsulatus was investigated. In the presence of uncouplers the rate of ATP hydrolysis was about 40 mM ATP/M bacteriochlorophyll (Bchl)/s. Without uncouplers this rate increased and if, additionally, the chromatophores were illuminated, it was almost doubled. If uncouplers were added shortly after illumination, the rate increased to 300-350 mM ATP/M Bchl/s. Obviously, energization of the membrane leads to the formation of a metastable, active state of the H(+)-ATPase. The maximal rate of ATP hydrolysis can be measured only when first all H(+)-ATPases are activated by delta mu H+ and when the delta mu H+ is abolished in order to release its back pressure on the hydrolysis rate. The half-life time of the metastable state in the absence of delta mu H+ is about 30 s. It is increased by 3 mM Pi to about 80 s and it is decreased by 1 mM ADP to about 15 s. Quantitatively, the fraction of active H(+)-ATPases shows a sigmoidal dependence on pHin (at constant pHout) and the magnitude of delta psi determines the maximal fraction of enzymes which can be activated: delta pH and delta psi are not equivalent for the activation process.

Adenine Nucleotides↗
PubMed1992

The structure of Photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals.

The structure of the Photosystem I (PS I) complex from the thermophilic cyanobacterium Synechococcus sp. has been investigated by electron microscopy and image analysis of two-dimensional crystals. Crystals were obtained from isolated PS I by removal of detergents with Bio-Beads. After negative staining, either single layers or two superimposed layers with a rotational different orientation were observed. The layers have a rectangular unit cell of 16.0 x 15.0 nm, which contains two PS I monomers. The monomers are arranged alternating up and down in each layer. For double-layer crystals, the images of the two layers could be separately processed by a combination of Fourier-peak-filtering and correlation averaging. Features in the two-dimensional plane can be seen with a resolution up to 1.5-1.8 nm. A model for the PS I structure was obtained by combining three-dimensional reconstructions from three tilt-series. The model shows an asymmetric PS I complex. On one side (presumably the stromal side) there is a 3 nm high ridge. This is most likely comprised of the psaC, psaD and psaE subunits. The other side (presumably the lumenal side) is rather flat, but in the center there is a 3 nm deep indentation, which possibly separates partly the two large subunits psaA and psaB.

Crystallization↗
PubMed1992

Proton transport-coupled unisite catalysis by the H(+)-ATPase from chloroplasts.

Proton transport-coupled unisite catalysis was measured with the H(+)-ATPase from chloroplasts. The reaction was measured in the ATP hydrolysis direction under deenergized conditions and in the ATP synthesis direction under energized conditions. The equilibrium constant of the enzyme does not change upon energization, whereas the dissociation constants of substrates and products change by orders of magnitude. This indicates that the Gibbs free enthalpy derived from proton translocation is used to change binding affinities of substrates and products, and this results in synthesis of free ATP.

Adenosine Triphosphate↗
PubMed1992

Numerical simulation of uni-site and bi-site ATP-hydrolysis catalyzed by the membrane-bound H(+)-ATPase from chloroplasts.

The kinetics of ATP-hydrolysis catalyzed by the H(+)-ATPase from chloroplasts was described by an enzyme kinetic model where either one (uni-site catalysis) or two (bi-site catalysis) sites are operating. Numerical simulations are carried out with the software package LARKIN using measured rate constants as a basis. When the ATP-concentration is increased up to 20 nM (at 20 nM enzyme concentration) a change from uni-site to bi-site catalysis occurs at about 7 nM. The measured effective rate constants change by a factor two. However, the calculated rate constants for the second site are drastically increased.

Adenosine Triphosphate↗
PubMed1992

Purification of the H(+)-ATPase from chloroplasts.

Triton X-100* was replaced in the purification of the H(+)-ATPase from chloroplasts by either octyl glucoside, octyl thio glucoside or the zwitterionic detergent CHAPS. The results indicate that the enzyme can be purified without Triton X-100 and retain full activity.

Chloroplasts↗
PubMed1991

The rate of ATP-synthesis as a function of delta pH and delta psi catalyzed by the active, reduced H(+)-ATPase from chloroplasts.

The H(+)-ATPase from chloroplasts was brought into the active, reduced state. Then, an electrochemical potential difference of protons across the thylakoid membranes was generated by an acid-base transition, delta pH, combined with a K+/valinomycin diffusion potential, delta psi. The initial rate of ATP synthesis was measured with a rapid-mixing quenched-flow apparatus in the time-range between 20-150 ms. The rate of ATP synthesis depends in a sigmoidal way on delta pH. Increasing diffusion potentials shifts the delta pH-dependencies to lower delta pH values. Analysis of the data indicate that the rate of ATP synthesis depends on the electrochemical potential difference of protons irrespective of the relative contribution of delta pH and delta psi.

Adenosine Triphosphate↗
PubMed1991

The plasma membrane H(+)-ATPase from yeast. Effects of pH, vanadate and erythrosine B on ATP hydrolysis and ATP binding.

The H(+)-ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated. The pH dependence of the rate of ATP hydrolysis forms a bell-shaped curve with a maximum at pH 6 and half-maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding. Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.

Adenosine Diphosphate↗
PubMed1991

ATP synthesis in chromatophores driven by artificially induced ion gradients.

An electrochemical potential difference for protons (delta mu H+) across the membrane of bacterial chromatophores was induced by an artificially generated pH difference (delta pH) and a K+/valinomycin diffusion potential, delta phi. The initial rate of ATP synthesis was measured with a rapid-mixing quenched-flow apparatus in the time range between 70 ms and 30 s after the acid-base transition. The rate of ATP synthesis depends exponentially on delta pH. Increasing diffusion potentials shift the delta pH dependency to lower delta pH values. Diffusion potentials were calculated from the Goldman equation. Using estimated permeability coefficients, the rate of ATP synthesis depends only on the electrochemical potential difference of protons irrespective of the relative contribution of delta pH and delta phi.

Adenosine Triphosphate↗
PubMed1990

Reconstitution of CF0F1 into liposomes using a new reconstitution procedure.

The H(+)-ATPase (ATP synthase) from chloroplasts was isolated, purified and reconstituted into phosphatidylcholine/phosphatidic-acid liposomes. Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100 and protein incorporation was studied at each step of the solubilization process. After detergent removal by SM2-Biobeads, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitution was obtained by insertion of the protein into preformed, detergent-saturated liposomes. The conditions for the reconstitution were optimized with regard to ATP synthesis driven by an artificially generated delta pH/delta psi. An important benefit of the new reconstituted CF0F1 liposomes is the finding that the rate of ATP synthesis remains constant up to 10 s, indicating a low basal membrane permeability.

Adenosine Triphosphate↗
PubMed1990

Uni-site ATP synthesis in thylakoids.

Uni-site ATP synthesis was measured with thylakoids. The membrane-bound ATP-synthase, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin, dithiothreitol and phosphate. This enzyme contains two tightly bound ATP per CF0F1. ATP was released from the enzyme when ADP was added in substoichiometric amounts during illumination. Experiments with [14C]ADP indicated that after binding the same nucleotide was phosphorylated and released as [14C]ATP, i.e. only one site is involved in ATP-synthesis ('uni-site ATP-synthesis'). The two tightly bound ATP are not involved in the catalytic turnover. The rate constant for ADP binding was (4 +/- 2) x 10(6) M-1s-1. Compared to deenergized conditions the rate constant for ADP binding and that for ATP-release were drastically increased, i.e. membrane energization increased the rate constants for the ATP-synthesis direction.

Adenosine Diphosphate↗
PubMed1990

Uni-site catalysis in thylakoids. The influence of membrane energization on ATP hydrolysis and ATP-Pi exchange.

ATP-hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme-bound ATP, ADP and Pi were measured using either cold ATP, [gamma-32P]ATP or [14C]ATP. The concentration of free ATP was constant, free ADP and enzyme-bound ATP were below the detection limit. Nevertheless, [gamma-32P]ATP was bound, hydrolyzed and 32Pi was released. The ADP was not released from the enzyme but cold Pi was bound from the medium, cold ATP was resynthesized and released. A quantitative analysis gave the following rate constants: ATP-binding kATP = 2 . 10(5) M-1 s-1, ADP-release: kADP less than 10(-2)s-1, Pi-release: kPi = 0.1 s-1. These rate constants are considerably smaller than under deenergized conditions. The rate constant for the release of ATP can be estimated to be at least 0.2 s-1 under energized conditions. Obviously, energization of the membrane, i.e. protonation of the enzyme leads mainly to a decrease of the rate of ATP-binding, to an increase of the rate of ATP release and to a decrease of the rate of ADP-release.

Adenosine Diphosphate↗
PubMed1990

The H(+)-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes.

The H(+)-ATPase of the plasma membrane from Saccharomyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H(+)-ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis-Menten kinetics with Km = 0.2 mM MgATP (plasma membranes), Km = 2.4 mM MgATP (isolated enzyme) and Km = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstitution; the activation of the H(+)-ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme-Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme-substrate complex, Ks, increases from Ks = 30 microM (plasma membranes) to Ks = 90 microM (isolated enzyme). ATP binding to the H(+)-ATPase ('single turnover' conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second-order rate constant k1 = 2.6 x 10(4) M-1.s-1. From these observations it is concluded that the detergent used (Zwittergent TM 3-14) interacts reversibly with the H(+)-ATPase and that practically all H(+)-ATPase molecules are reconstituted into the liposomes with the ATP-binding site being directed to the outside of the vesicle.

Adenosine Triphosphate↗
PubMed1990

Activation/inactivation and uni-site catalysis by the reconstituted ATP-synthase from chloroplasts.

The proton-translocating ATP-synthase of chloroplasts, CF0F1, was isolated and reconstituted into asolectin liposomes. CF0F1 can exist in at least four different states, oxidized or reduced, either inactive or active. These states are characterized by different kinetics of ADP binding: There is no binding of ADP to the inactive, oxidized state, the rate constant for ADP binding to the inactive, reduced states is 7.10(2) M-1.s-1. ADP binding to the active, reduced state occurs under deenergized conditions with 10(5) M-1.s-1 and transforms the enzyme into the inactive, reduced state. Parallel to the ADP-dependent inactivation, the enzyme can also inactivate without ADP binding with a first-order rate constant of 7.10(-3) M-1.s-1. With the active, reduced enzyme ATP-hydrolysis was measured under uni-site conditions as has been carried out with MF1 (Grubmeyer, C., Cross, R.C. and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100). The rate constant for ATP binding is 10(6) M-1.s-1, the 'equilibrium constant' on the enzyme EADPPi/EATP is 0.4. The rate constants for Pi release and ADP release are 0.2 s-1 and o.1 s-1, respectively. This indicates that the enzyme carries out a complete turnover under uni-site conditions with rates much higher than that reported for MF1.

Adenosine Diphosphate↗
PubMed1979

The plastoquinone pool as possible hydrogen pump in photosynthesis.

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H+ + e-) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H+ uptake and the internal H+ release led to the following conclusions: (1) A stoichiometric coupling of 1 : 1 : 1 between the external H+ uptake, the electron translocation through the plastoquinone pool and the internal H+ release (corrected for H+ release due to H2O oxidation) is valid (pHout = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation. These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H+ + e-) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.

Biological Transport, Active↗