pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803.

It was shown before (Wooten, D. C., and Dilley, R.

Introduction of a carboxyl group in the loop of the F0 c-subunit affects the H+/ATP coupling ratio of the ATP synthase from Synechocystis 6803.

The proton translocation stoichiometry (H+/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H+/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (deltaGp) and the proton gradient (deltamuH+) induced by acid-base transition.

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase.

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency.

The ATP synthase gamma subunit provides the primary site of activation of the chloroplast enzyme: experiments with a chloroplast-like Synechocystis 6803 mutant.

The activation characteristics of the F1Fo-ATP synthase (where F1 and Fo are the hydrophilic and membrane-bound parts respectively of the enzyme) from Synechocystis 6803 wild-type and a Synechocystis 6803 mutant with a chloroplast-like insertion in the gamma subunit have been studied. Activation of the ATP synthase in wild-type and mutant membrane vesicles was performed by acid-base transition-induced generation of a proton motive force (Delta mu H+). Since the mutant containing the regulatory segment of the chloroplast gamma subunit showed thiol-modulation (typical of the chloroplast enzyme), this segment is indeed involved in the regulation of enzyme activation.

The effect of sulfite on the ATP hydrolysis and synthesis activities in chloroplasts and cyanobacterial membrane vesicles can be explained by competition with phosphate.

The effect of sulfite on ATP synthesis and hydrolysis activities is investigated in spinach chloroplasts and in membrane vesicles from the cyanobacterium Synechococcus 6716. Sulfite inhibits phenazine methosulfate-mediated cyclic photophosphorylation both in thiol-modulated chloroplasts and in cyanobacterial membranes with HSO3- (bisulfite) as the active ionic species. The observed inhibition is not due to inhibition of electron transfer or to uncoupling by sulfite.

Membrane vesicles fromSynechocystis 6803 showing proton and electron transport and high ATP synthase activities.

A simple procedure for the preparation of well-coupled and stable membrane vesicles from the transformable cyanobacteriumSynechocystis 6803 is described with the primary aim of producing vesicles suitable for the study of photosynthetic electron transport and phosphorylation. Spheroplasts were obtained from the cyanobacterium by lysozyme treatment and stored untill prior to measurement, thylakoid vesicles were obtained by osmotic shock. These vesicles showed very high and stable ATP synthesis rates either driven by light or by acid-base transition, and also performed light-induced ATP hydrolysis and linear electron transport.

The H+/ATP coupling ratio of the ATP synthase from thiol-modulated chloroplasts and two cyanobacterial strains is four.

In this paper the authors emphasise that the proton translocating ATP synthase from thiol-modulated chloroplasts and two cyanobacterial strains has a coupling ratio of 4 protons per ATP synthesised or hydrolysed. This ratio is determined by several thermodynamic studies at equilibrium between phosphate potential (Delta Gp) and proton gradient (Delta(mu)H+), and is confirmed by measurement of proton flux during ATP hydrolysis. Ratios lower than 4 H+/ATP that have been published in the past have predominantly been determined with the oxidised chloroplast enzyme.

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.

Potassium selective and venturicidin sensitive conductances of Fo purified from bovine heart mitochondria, reconstituted in planar lipid bilayers.

Purified F(o), isolated from bovine heart mitochondria was reconstituted into planar lipid bilayers. Two cation selective conductances could be identified. Most frequently, incorporation of F(o) resulted in a voltage sensitive K+ channel of 18 pS.

The effect of sulfite on the ATP hydrolysis and synthesis activity of membrane-bound H(+)-ATP synthase from various species.

The action of sulfite on ATP hydrolysis and synthesis activities is investigated in membrane vesicles prepared from the cyanobacterium Synechococcus 6716, chromatophores from the photosynthetic purple bacterium Rhodospirillum rubrum, membrane vesicles from the related non-photosynthetic bacterium Paracoccus denitrificans, and bovine heart submitochondrial particles. Without any further pretreatment ATP hydrolysis is stimulated by sulfite in all four membrane preparations. Typically ATP synthesis in the cyanobacterial membrane vesicles is inhibited by sulfite, whereas ATP synthesis in chromatophores and the submitochondrial particles is not.

F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function.

Modulation of the proton-translocation stoichiometry of H(+)-ATP synthases in two phototrophic prokaryotes by external pH.

The stoichiometry between proton translocation and ATP synthesis/hydrolysis was studied in two different photosynthetic prokaryotes, the thermophilic cyanobacterium Synechococcus 6716 and the purple bacterium Rhodospirillum rubrum. The H+/ATP ratio was determined by acid-base transitions as a function of the external pH. The H+/ATP ratio of the Synechococcus 6716 ATP synthase was found to increase with increasing pH.

Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716.

The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha, beta, delta and epsilon, by determining their N-terminal sequences. They are arranged at three separate loci.

On the activation mechanism of the H(+)-ATP synthase and unusual thermodynamic properties in the alkalophilic cyanobacterium Spirulina platensis.

The activation requirements and thermodynamic characteristics of ATP synthase from the alkalophilic cyanobacterium Spirulina platensis were studied in coupled membrane vesicles. Activation by methanol increased the Vmax, while the Km for MgATP was unaffected (0.7 mM).

Effect of Elicitors on the Plasmamembrane of Petunia hybrida Cell Suspensions : Role of DeltapH in Signal Transduction.

Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia lyase activity.

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 wavelength 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.

Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes.

The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated. The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain. Some commercial lipids were used as well.

Proton movements and electric potential generation in reconstituted ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716.

ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur.

Purification of a high molecular weight membrane protein by fast protein liquid chromatography: the ATPase complex of a thermophilic cyanobacterium.

Fast protein liquid chromatography (FPLC) with a strong anion-exchange (Mono Q) column is applied to the purification of a high molecular weight membrane protein. The ATPase complex of the thermophilic cyanobacterium Synechococcus 6716, partially purified by ammonium sulfate precipitation, was fractionated in the presence of the detergent octylglucoside. The ATPase complex containing fractions were eluted by a linear NaCl gradient at about 0.

Thermotolerance in cultured hepatoma cells: cell viability, cell morphology, protein synthesis, and heat-shock proteins.

Heat treatment at 42 degrees C of cultured Reuber H35 rat hepatoma cells induced both a rapid decrease of the rate of protein synthesis and the rounding up of the cells. Reincubation at 37 degrees C resulted in a gradual flattening of the cells, resumption of protein synthesis, and the synthesis of heat-shock proteins. During the recovery period cells developed a resistance toward a treatment which otherwise should lead to heat-induced cell death.

Isolation, purification and characterization of the ATPase complex from the thermophilic cyanobacterium Synechococcus 6716.

The ATPase complex is isolated and purified from membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 by octyl glucoside and cholic acid by a modification of the procedure for its extraction from spinach chloroplasts. The complex is purified by differential centrifugation and ammonium sulfate precipitation and by gel filtration on Sepharose 6B. The purified fraction, without any phycocyanin contamination, shows ATP hydrolysis activity and Pi/ATP exchange activity of 1564 and 350 nmol X min-1 X mg protein-1, respectively.

Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716.

The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716. In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze-fracture replicas.