H.S. van Walraven

Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study)

In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

Dependence of the proton translocation stoichiometry of cyanobacterial and chloroplast H+-ATP synthase on the membrane composition

Abstract The high proton translocation stoichiometry (approx. 9 H+ / ATP) of ATPase proteoliposomes reconstituted from a thermophilic cyanobacterium (Van Walraven et al. (1986) FEBS Lett. 208, 138–142) has also been observed with chloroplast ATP synthase when reconstituted with cyanobacterial lipids. Both enzyme complexes in isolated and reconstituted form show highest stable trypsin-activated ATP hydrolysis activity at the same temperature (55°C). Also, both isolated ATP synthases require the same reconstitution procedure for maximal coupling quality. The proton translocation stoichiometry has been deduced from the relation between the initial rates of ATP hydrolysis at varying sizes of the electrochemical potential gradient ( Δ \ gm H + ). A Δ \ gm H + was imposed by valinomycin-induced K+ diffusion potentials or by base-pulses which were equally efficient in inhibiting ATP hydrolysis. Kinetic experiments with the use of the pH indicator Cresol red confirm the high proton translocation stoichiometry of both types of ATPase proteoliposome. Functional co-reconstitution of both types of ATPase proteoliposome with cyanobacterial cytochrome b6f complex leads to a decrease in proton translocation stoichiometry to about 7 H+ / ATP. Cyanobacterial membrane vesicles take up 4.4 protons per ATP hydrolyzed. A value of 4.5 H+ / ATP is observed with chloroplasts in equilibrium (Graber, P., Junesch, U. and Thulke, G. (1986) in Progress in Photosynthesis Research (Biggins, J., ed.), pp. 177–184, Martinus Nijhoff, Dordrecht). These results indicate that the proton translocation stoichiometry of the ATP synthase depends on the membrane composition. The consequence of this finding for the mechanism of proton translocation and the possible physiological relevance are discussed.

ATP synthesis by ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716 by ionophore-induced electric potentials and proton gradients

Abstract ATP synthesis driven by low pre-established electric potentials and pH gradients is studied in large ATPase proteoliposomes, prepared from the ATPase complex and native lipids from the thermophilic cyanobacterium Synechococcus 6716. Electric potentials and pH gradients were achieved by valinomycin and nigericin, respectively, in the presence of a K + gradient across the membrane. External base-pulses were also applied. In this system ATP synthesis driven by valinomycin-induced K + influx, nigericin-induced internal acidification and by external base-pulses is demonstrated. Electric potentials and pH gradients of equivalent size lead to roughly similar ATP synthesis activities. ATP synthesis is optimal at 80–100 nM valinomycin and at 0.75−1 μM nigericin at the proper pre-set ion gradients. Uncoupler and DCCD inhibit ATP synthesis. Prior activation of the complex by thiol agents or trypsin was not required for synthesis activity. The ATP synthesis rate increases with the size of electric potential or pH gradient. The threshold value of the electrochemical gradient for significant ATP synthesis is about 30 mV. ATP production proceeds for more than 60 min. The generation of ionophore-induced electric potentials and pH gradients have been followed by oxonol VI and intraliposomal Neutral red, respectively. The extent of the absorbance changes of both probes is proportional to the size of electric potential or pH gradient. Ionophore-induced oxonol VI and Neutral red responses are stable for at least 30 min. The results are discussed in terms of membrane permeability and vesicle size.

Activation of the H+-ATP synthases of a thermophilic cyanobacterium and chloroplasts — a comparative study

The activation requirements of the ATP synthases of the thermophilic cyanobacterium Synechococcus 6716, studied in coupled membrane vesicles and in the isolated or reconstituted complex, and of the ATP synthase of spinach chloroplasts were compared. It was found that methanol, heat treatment or dithiothreitol did not activate ATP hydrolysis in Synechococcus 6716. In contrast to the chloroplast enzyme, activation could only be accomplished with sulfite, octyl glucoside, a proton electrochemical potential difference and trypsin. The lack of activation by dithiothreitol, heat and methanol in the cyanobacterial ATP synthase can be explained by the absence of three cysteine residues in the regulatory γ subunit of the F 1 part. The threshold value of the proton electrochemical potential difference at which ATP synthesis occurs at low G p was about 9.5 kJ mol −1 for the cyanobacterial ATP synthase. This is similar to the threshold value of the reduced form of the enzyme in chloroplasts. With cyanobacterial membrane vesicles, an H + / ATP stoichiometry slightly exceeding 4 was obtained in ATP hydrolysis as well as in ATP synthesis, measured as a function of an artificially applied proton electrochemical potential difference. These findings are discussed in terms of a single structural difference between the cyanobacterial and the chloroplast enzyme. When comparing the enzyme of Synechococcus 6716 with that of chloroplasts, our results indicate that the difference in activation requirements of both ATP synthases resides in a different arrangement of the γ and e subunits.

The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans‐membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelenght is obtained with valinomycin‐induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.

The significance of interfacial charge and proton displacements for the mechanism of energy transduction in biomembranes

Abstract Energization of chloroplast membranes induces a significant increase of negative surface charge density, as appears from measurements of the electrokinetic potential and the adsorption of cationic probes. This phenomenon is correlated with the displacement of protons from the external interfacial layer. The estimations of the interfacial potential were compared to the actual surface potential of liposomes with known surface charge density, and large discrepancies were apparent. Transient interfacial pH changes were observed in subchloroplast vesicles in response to single-turnover energization by short light flashes. Reconstituted ATPase proteoliposomes with internally trapped and externally added pH indicators were used for kinetic analysis of the sequential steps of ATP-driven proton influx. A clear discrimination of external and internal, as well as interfacial and bulk proton displacements, was possible. These findings are accommodated in a simple model for energy-linked proton displacements and changes of surface charge, which takes into account the significance of both localized and delocalized charge reorientations and proton gradients.

Activation of the H+-ATP synthase in thylakoid vesicles from the cyanobacterium Synechococcus 6716 by Δ\̄gmH+. Including a comparison with chloroplasts, and introducing a new method to calibrate light-induced Δ\̄gmH+

Abstract Activation of ATP hydrolysis activity in well-coupled membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 has been studied. In addition to a basal (dark) activity dependent on culture age, the cyanobacterial thylakoid ATP synthase is activated by Δ \ gm H + . Activation of this enzyme has been studied quantitatively and the results are compared with literature data regarding activation of the chloroplast ATP synthase. We found that activation behaviour both in chloroplasts and in cyanobacterial membrane vesicles may be characterized by the value of Δ \ gm H + where 50% activation occurs ( Δ \ gm a ) and a Hill-type coefficient (p) that estimates the number of protons involved. The activating Δ \ gm H + was induced either by the acid-base pulse method, or by illumination. To calibrate the size of light-induced Δ \ gm H + , a method has been developed whereby at each light intensity the value of ΔGp is determined where the ATP synthase reaction is in equilibrium. Δ \ gm H + at equilibrium then is calculated from this ΔGp and the H + ATP ratio. The value of p for the cyanobacterial ATP synthase is similar to that of the chloroplast enzyme (between 1 and 2). The cyanobacterial enzyme resembles the thiol-modulated (reduced) form of the chloroplast ATP synthase in that Δ \ gm a is low (14.1 kJ mol−1) and that the Δ \ gm H + -activated state is quite stable. As in the thiol-modulated chloroplast ATP synthase, decay of the activated state is accelerated by ADP. We conclude that the Synechococcus 6716 membrane vesicles are an excellent system in which to study Δ \ gm H + activation of ATP synthase with no need for thiol modulation.

Measurement of diffusion potentials in liposomes. Origin and properties of the threshold level in the oxonol VI response

Abstract A model is presented for the response of the membrane potential probe oxonol VI on diffusion potentials in liposomes. In this model the dependence of the probe response on the initial ion gradient is explained in terms of internal volume, internal ion concentration, membrane capacity and initial membrane potential. It is found that in the presence of an initial membrane potential (positive outside) there is a threshold value of the ion gradient needed for a probe response, which increases when the internal volume or the internal ion concentration decrease. The model is confirmed by experiments with liposomes of different sizes and internal KCl concentrations, prepared from asolectin or lipids isolated from the thermophilic cyanobacterium Synechococcus 6716. The significance of the model for threshold values observed in other energy-dependent phenomena is discussed.

Comparison of ATP synthesis efficiencies in ATPase proteoliposomes of different complexities

Abstract ATP synthesis by the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 has been studied in each of three reconstituted proteoliposome systems: • ATPase complex reconstituted with the native lipids of Synechococcus 6716 by detergent dialysis. ATP synthesis of about 100 nmol min−1 (mg protein)−1 was observed at 50 ° C after a valinomycin-induced K+ diffusion potential (inside positive), nigericin-induced K+-H+ exchange (internal pH decrease) and an external base pulse, equivalent to about 65 mV. • ATPase complex co-reconstituted with bacteriorhodopsin of Halobacterium halobium and soybean phospholipids by sonication and detergent removal by gel filtration. Light-induced ATP synthesis at 40 ° C of 70 nmol min−1 (mg ATPase protein)−1 was obtained. Absorbance changes of the electric potential-indicating probe oxonol VI induced by flashed and continuous light were observed. In this preparation, with the pH indicator neutral red, only light-driven external alkalinization could be detected. • ATPase complex co-reconstituted by detergent dialysis with bovine-heart ubiquinol: cytochrome c oxidoreductase and photosynthetic reaction centres of Rhodopseudomonas sphaeroides and soybean phospholipids, with intraliposomal cytochrome c and additional Q-10. Light-driven synthesis of about 1.5 nmol min−1 (mg ATPase protein)−1 was obtained at 40°C. Flash-induced internal cytochrome c reduction was also demonstrated in this preparation.

Evidence for a high proton translocation stoichiometry of the H+-ATPase complex in well coupled proteoliposomes reconstituted from a thermophilic cyanobacterium

Evidence is presented for a high proton translocation stoichiometry (H+/ATP) of approx. 9 in ATPase proteoliposomes with extremely low permeability for ions, reconstituted from a thermophilic cyanobacterium. A proportional relation between the phosphate potential (ΔG fp) and the proton‐motive force (Δp) was observed in thermodynamic equilibrium. A bulk‐to‐bulk Δp was imposed by valinomycin‐induced K− diffusion potentials of different size while the initial ΔG fp was varied. In all cases equilibrium was reached in about 1.5 h. A high H−/ATP ratio was also deduced from the relation between the initial rates of ATP synthesis or hydrolysis at varying ΔG fp and Δp. The implications of these results for the mechanism of energy transduction in energy‐conserving membranes are discussed.