Extraction of chromatophores from Rhodospirillum rubrum in aqueous two-phase systems: A method for large-scale isolation of membrane particles

Chromatophores, membrane vesicles with the capacity of cyclic photophosphorylation, have been isolated from Rhodospirillum rubrum cells on a pilot plant scale. Results of disintegration in a glass bead mill and in a high pressure homogenizer were compared. The chromatophores were isolated from the crude extract by extraction in aqueous two-phase systems. In systems of polyethylene glycol (PEG) and dextran the chromatophores were partitioned to the upper PEG phase by the addition of PEG-palmitate. Most of the proteins and nucleic acids were forced to the bottom phase by addition of sodium chloride. Methods to prevent precipitation of the chromatophores were studied.     

Activation-deactivation reactions in the ATPase enzyme in Rhodospirillum rubrum chromatophores

(1) The ATPase enzyme in untreated chromatophores from Rhodospirillum rubrum is in a low-activity state (designated as E°). It can be activated by application of a transmembrane generated by light-induced electron transport, or by application of acid-base jumps. (2) After rapid dissipation of the light-induced , the active state of the ATPase enzyme decays (in the absence of added substrates or products) to a low-activity state (designated as E′), with a half-time of the order of 2–4 min. This state differs from E° in that E′ (but not E°) can be rapidly reactivated by addition of substrate, but only when the Mg²⁺ concentration is kept below 20–30 μM. Since this is characteristic of an activated enzyme containing tightly bound ADP (Slooten, L. and Nuyten, A. (1981) Biochim. Biophys. Acta 638, 313–326), it is suggested that release of endogenous, tightly bound ADP is one of the factors involved in activation of the ATPase enzyme.     

Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores

(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg²⁺). I₅₀ values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min^?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg²⁺ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg²⁺ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg²⁺. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg²⁺ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg²⁺ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.     

Regulation of cyclic photophosphorylation in Rhodospirillum rubrum by the redox state of nicotinamide-adenine dinucleotide

We have investigated the effect of the redox state of added NAD on the rates of anaerobic cyclic photophosphorylation which are supported by membrane vesicles isolated from Rhodospirillum rubrum. As the redox potential of NAD was lowered, the activity decreased according to a typical potentiometric titration. The Nernst plot showed an apparent midpoint potential (E′_o) of ?350 mV and had a slope which corresponded to a two-electron transition. Besides, an almost identical potentiometric relationship was found to exist between the extent of light-elicited ATP formation in anaerobic suspensions of intact R. rubrum cells and the redox potential of intracellular NAD. These results suggest that physiological photophosphorylation in R. rubrum requires the oxidized form of a membrane-bound constituent (E′_o = ?350 mV) whose redox state is controlled by the redox state of cytoplasmic NAD.     

Kinetics of the membrane-bound inorganic pyrophosphatase from Rhodospirillum rubrum chromatophores: Effect of the transmembrane electrical potential on the rate constants

The behaviour of the membrane-bound proton-translocating pyrophosphatase (H⁺-PPase) in Rhodospirillum rubrum chromatophores upon application of an electrochemical potential is studied. The rate constants are shown to be affected in an asymmetric fashion. The forward rate constant (PP_i synthesis) is shown to be at least 45-times larger during illumination than when there is no proton-motive force. The hydrolysis rate is increased maximally 8-times when the potential is dissipated. The effect of the electrical field gradient is thus mainly to increase the forward rate of the reaction. The H⁺-PPase also seems to be a functionally simpler enzyme than the H⁺-ATPase, lacking the hydrolysis activation step during energization found in the latter.     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

Exogenous cytochrome c-dependent light-induced membrane potential generation in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores associated with a planar phospholipid macromembrane by bivalent cations in the presence of quinone, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and ascorbate generate a transmembrane electrical potential difference in the light. Photoelectrical activity is also observed if chromatophores are preincubated with cytochrome c; the maximum values of responses are reached upon subsequent addition of ascorbate and menadion in the absence of bivalent cations and TMPD. The cytochrome c-dependent responses of the illuminated chromatophores are inhibited by Ca²⁺ and prevented by quinones. The possibility of cytochrome c (c₂) translocation across the chromatophore membrane and the mechanism of charge transfer across the planar phospholipid membrane are discussed.     

Regulation of the plasma membrane potential in Pneumocystis carinii

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.     

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

盐胁迫对小麦根质膜ATPase活性的影响

以小麦为实验材料,研究了盐胁迫对根质膜H^ —ATPase、Ca^2 —ATPase活性及H^ —ATPase蛋白表达的影响。结果显示：50、100、150mmol/L的NaCl处理72h后,小麦根质膜H^ —ATPase、Ca^2 —ATPase活性均降低。100mmol／L NaCl对质膜ATPase活性的抑制程度随处理时间的延长而增强,在处理24h后,H^ —ATPase和Ca^2 —ATPase的活性分别降为对照的72％和75％,而处理72h后,酶活性分别减小到对照的50％和48％。50、100、150mmol／L的NaCl直接作用于提取的质膜微囊,H^ —ATPase的活性分别降低约5％、8％和16％。Western blotting分析结果显示100mmol／L NaCl处理72h后,质膜H^ —ATPase的含量与对照比有所减少。本研究表明：盐胁迫抑制小麦根质膜H^ —ATPase、Ca^2 —ATPase的活性,酶含量的减少可能是盐胁迫导致质膜H^ —ATPase活性降低的原因。     

Effect of potassium deficiency on the plasma membrane H+-ATPase of the wood ray parenchyma in poplar

The effect of K⁺ deficiency on the plasma membrane (PM) H⁺‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K⁺ supply. Immunological assays using different antibodies were applied to test if K⁺ deficiency affects the amount of immunodetectable PM H⁺‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H⁺‐ATPases under conditions of low K⁺ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H⁺‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K⁺ supply. Measurements of extracellular H⁺ concentrations using non‐invasive, H⁺‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H⁺ in plants grown with low K⁺ supply. The present results indicate an up‐regulation of specific isoforms of the PM H⁺‐ATPase in VACs under K⁺‐deficient conditions and suggest a key role for these PM H⁺‐ATPases in unloading K⁺ from the xylem stream.     

Evidence for regulation in vivo of the ATP synthase in intact cells of the photosynthetic bacterium Rhodopseudomonas capsulata

(1) The cytoplasmic membrane potential (Δψ) of intact cells of Rhodopseudomonas capsulata, measured either from the uptake of butyltriphenylphosphonium cation or from the electrochromic carotenoid band shift, increased upon illumination (negative on the cytoplasmic side) and then, within the next 20 s, partly declined while the light was still on. In the presence of the F₀ inhibitor venturicidin the light-induced Δψ was increased by 30% and the partial decline was abolished. (2) From the ionic current/Δψ curves for the bacterial membranes it was concluded that the slow, partial decline of Δψ after the onset of illumination was the result of an increase in membrane conductance. The conductance increase seen in the ionic current/Δψ curves was blocked by venturicidin suggesting that it was caused by increased proton flux through the ATP synthase. (3) Analysis of the light-induced changes in adenine nucleotide levels in intact bacterial cells showed that the apparent increase in ATP synthase activity was not the result of a decrease in phosphorylation potential. The data were consistent with either an increase in the catalytic activity of the ATP synthase or with an increase in H⁺ flux through the enzyme without a proportionate increase in the rate of phosphorylation (increased ‘slip’). (4) This slow change in the properties of the ATP synthase, as judged by the venturicidin-sensitive partial decline of Δψ, required a minimum initial value of Δψ. When Δψ was reduced, either by decreasing the actinic light intensity or by adding carbonylcyanide trifluoromethoxyphenylhydrazone the partial decline in Δψ was abolished. (5) The slow change in ATP synthase properties reversed upon darkening the bacterial cell suspension. A second illumination period shortly after the first elicited a smaller initial Δψ and a smaller Δψ decline. The relaxation of the ATP synthase in the dark was measured from the dependence of the initial increase in Δψ after the second illumination period upon the dark-time between the two illumination periods.     

Influence of ATPase activity on PPi dependent H-transport in tonoplast vesicles of Acer pseudoplatanus

Tonoplast H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H⁺-transports were also additive. The H⁺ -PPase inhibition did not change ATP-dependent H⁺-transport but H⁺-ATPase inhibition inhibited the PPi dependent H⁺-transport. Because H⁺-PPase was reported to transport H⁺ and K⁺ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H⁺-ATPase activity could modify the H⁺/K⁺ stoichiometry for the benefit of K⁺-transport.     

Kinetics of the purified plasma membrane H+-ATPase from red beet (Beta vulgaris)

The plasma membrane H⁺-ATPase (EC 3.6.1.35) was purified by washing red beet ( Beta vulgaris L.) plasma membranes with sodium deoxycholate and separating the ATPase, solubilized with lysophosphatidylcholine, by centrifugation in a glycerol gradient. The purified H⁺-ATPase had a sedimentation coefficient of about 8S. In the absence of exogenous protein substrates, the purified ATPase preparation did not present protein kinase activity. Compared with the H⁺-ATPase in the plasma membrane, the purified ATPase presented a higher affinity for adenosine 5'-triphosphate (ATP) and a lower sensitivity to the inhibitors vanadate and inorganic phosphate. These changes in the kinetics of the ATPase could also be observed by treating the membranes with lysophosphatidylcholine, without purifying the enzyme. These results can be explained assuming that lysophosphatidylcholine interacts with the ATPase altering its kinetics probably by stimulating the transformation from the inhibitor-binding conformation E2 into the ATP-binding conformation E1.     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

Schistosoma mansoni: Characterization of the electrical potential from the tegument of adult males

An electrical potential having a value of ?35 mV (SD = 7.4) is recorded upon penetration of the ventral surface of adult male Schistosoma mansoni with a microelectrode. Histological studies using horseradish peroxidase as a marker injected iontophoretically through the recording electrode indicate that this potential originates in the tegumental epithelium. Recordings from animals with contractile activity showed spontaneous nonovershooting depolarizations with durations of 20 to 100 msec and amplitudes between 5 and 15 mV. Slow-wave depolarizations are also observed and are most frequent in animals showing only slight movement. The tegumental membrane potential appears to be dependent primarily on the K⁺ gradient across the surface since a 10-fold increase in external K⁺ causes a 30-mV change in the potential. Altering the external concentration of Cl^? has little effect on the tegumental potential, but lowering the external Na⁺ concentration to 37 mM causes a 10-mV depolarization. Praziquantel (PZ) has little initial effect on the tegumental membrane potential. A slow depolarization does occur, however, which reaches a significant level about 10 min after PZ (1 μM). Carbachol and dopamine are without significant effects on the tegumental membrane potential.     

H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.