Kinetic characterization of the ATP-dependent proton pump in bacterial photosynthetic membranes: a study with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine

Fregni, Valeria; Casadio, Rita

doi:10.1016/0005-2728(93)90146-7

Cited by 13 publications

(7 citation statements)

References 34 publications

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“…The results of this work demonstrate that the hydrolytic activity of the ATP synthase in R. cupsulutus can be strongly activated by sulfite. This observation extends previous studies in other organisms, such as other Rhodospirillaceae (Webster et al, 1977;Fregni and Casadio, 1993), archebacteria (Schobert, 1991(Schobert, , 1993 cyanobacteria (Bakels et al, 1994), higher plant choloroplaats Du and Boyer, 1990) and mitochondria (Moyle and Mitchell, 1975 ;Vasilyeva et a]., 1982;Cernyak et al, 1988). The activation by sulfite is therefore a general feature of all ATP synthases tested.…”

Section: Discussionsupporting

confidence: 87%

“…We demonstrate here that the sulfite-activated state is unable to translocate protons across the membrane or to promote chemiosmotic ATP synthesis and that the mechanism of sulfite activation involves probably two binding sites, with which phosphate binding can interfere. This work confirms and expands previous data from our laboratory demonstrating the concomitant stimulation of hydrolysis and of ATP-driven proton translocation by submillimolar concentrations of sulfite (Fregni and Casadio, 1993).…”

supporting

confidence: 91%

“…In the absence of autoactivation these concentrations would have caused only a very small stimulation of the rate of hydrolysis coupled to proton translocations (Fig. 4;Fregni and Casadio, 1993). At high concentrations of sulfite an increase in the rate was observed, reaching values above those of controls and similar to those shown in Fig.…”

Section: Resultssupporting

confidence: 62%

“…The reaction was initiated by the addition of equimolar concentrations of ATP and MgCl, in the range 0.1-2 mM; the concentration of Mg" endogenous in the chromatophore preparation was measured colorirnetrically (Orange and Rhein, 1951) and was included in the calculation of the overall concentration. The concentration of MgATP2-indicated in the figures, were calculated considering the multiple equilibria of binding between the cations, nucleotides and other anions present in the assay, particularly the sulfite dianion (Fregni and Casadio, 1993). Alternatively the ATP hydrolysis activity was measured continuously with a glass electrode (Schott type S218), measuring the scalar protons produced by the hydrolysis reaction at alkaline pH (Nishimura et al, 1962;Turina et al, 1992), using a Radiometer pHMeter (Model PH26) equipped with an electronic filter and offset voltage.…”

Section: Methodsmentioning

confidence: 99%

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Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by Δ_H+

Cappellini

Turina

Fregni

et al. 1997

European Journal of Biochemistry

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Sulfite stimulates the rate of ATP hydrolysis by the ATP synthase in chromatophores of Rhodobucter capsulatus. The stimulated activity is inhibited by oligomycin. The activation takes place also in uncoupled chromatophores. The activation consists in an increase of about 12-15-fold of the V,,,,, for the ATP hydrolysis reaction, while the K,,, for MgATP is unaffected at 0.16i0.03 mM. The dependence of V,,,,, on the sulfite concentration follows a hyperbolic pattern with half maximum effect at 12 mM.Sulfite affects the ability of the enzyme in translocating protons. Concomitant measurements of the rate of ATP hydrolysis and of ATP-induced protonic tlows demonstrate that at sulfite concentrations of greater than 10 mM the hydrolytic reaction becomes progressively uncoupled from the process of proton translocation. This is accompanied by an inhibition of ATP synthesis, either driven by light or by artificially induced ionic gradients. ATP synthesis is totally inhibited at concentrations of at least 80 mM.Sulfite interferes with the mechanism of activation by d P H , . Low concentrations of this anion ( S 2 mM) prevent the activation by A$,,+. At higher concentrations a marked stimulation of the activity prevails, regardless of the occurrence of a AFHt across the membrane. Phosphate at millimolar concentrations can reverse the inhibition by sulfite.These experimental results can be simulated by a model assuming multiple and competitive equilibria for phosphate or sulfite binding with two binding sites for the two ligands (for sulfite K,, = 0.26 and K2? = 37 mM, and for phosphate K , , = 0.06 and K2p = 4.22 mM), and in which the state bound only to one sulfite molecule is totally inactive in hydrolysis. The competition between phosphate and sulfite is igands and of the enzyme.ATP-driven proton translocation; protonic slipping; consistent with the molecular structures of the two

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Section: Discussionsupporting

confidence: 87%

supporting

confidence: 91%

Section: Resultssupporting

confidence: 62%

Section: Methodsmentioning

confidence: 99%

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Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by Δ_H+

Cappellini

Turina

Fregni

et al. 1997

European Journal of Biochemistry

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“…A theoretical justification of the use of fluorescence probes for kinetic measurements has been published (121, and computer analysis of the quenching of the fluorescence of probes such as acridine orange has been used to quantify H' fluxes [24]. ACMA has been shown to be the probe of choice for the measurement of relatively small dpH values [25], and we have shown previously [I41 both that the response of ACMA fluorescence to changes in ApH in ATPase proteoliposomes is very rapid, and that the rate of fluorecence quenching is proportional to reaction rate over a very wide range.…”

Section: Discussionmentioning

confidence: 99%

Allosteric Regulation of Proton Translocation by a Vacuolar Adenosinetriphosphatase

Webster

Perez-Castiñeira

Atkins

et al. 1995

European Journal of Biochemistry

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The kinetics of nucleoside-triphosphate-dependent proton translocation by a vacuolar-type adenosinetriphosphatase have been studied, using the enzyme from bovine chromaffin-granule membranes, purified and reconstituted into proteoliposomes. The reaction was followed by recording the quenching of the fluorescence of the permeant weak base 9-amino-6-chloro-2-methoxyacridine; fluorescence data were collected and stored in digital form, and the initial reaction rates estimated by linear regression. In the absence of nucleoside diphosphate, the dependence of initial rates of proton translocation on substrate concentration were fitted well by the Michaelis-Menten equation, as were the kinetics of ATP hydrolysis.ADP and other nucleoside diphosphates were potent inhibitors of the ATPase, effecting a reduction in the maximum velocity of the reaction, and producing sigmoid substrate-saturation curves which could be fitted by the empirical Hill equation, the Hill coefficient approaching 2 at high inhibitor concentrations. Data sets containing initial-rate estimates were collected over a wide range of independently varied concentrations of substrate and inhibitor and were modeled, using rate equations derived from several different models based on the concerted-transition model of allosteric inhibition proposed by Monod, Wyman and Changeux. These equations were fitted to the data by weighted non-linear regression, using an iterative computer program to obtain the best estimates of kinetic parameters. One model consistently fitted all of the data sets better than all the others, and this model was based on the following assumptions:that the ATPase exists in two conformational states, R and T ; that only the R state is catalytically active; that each state contains three kinetically equivalent catalytic sites, and one regulatory site; that nucleoside triphosphates bind only to the catalytic sites, and that nucleoside diphosphates bind both to the catalytic sites and to the regulatory site. The optimized values of the kinetic parameters indicate that in the absence of nucleoside diphosphate, the enzyme is almost completely in the R state ; that nucleoside triphosphates bind more tightly to the R than to the T state; that binding of nucleoside diphosphates to the regulatory site is very tight, but occurs only in the T state; and that competitive binding of nucleoside diphosphates at the catalytic sites is stronger in the T state than in the R state. Experiments conducted with varying total magnesium concentrations indicated that the magnesium complexes of nucleoside diphosphates are much stronger inhibitors than the free nucleotides, and that free nucleoside triphosphates are weakly inhibitory, probably competing with the magnesium complexes for binding at the catalytic sites. The results of these experiments indicate that the effects of nucleoside diphosphates, particularly ADP, occur at concentration ranges that are likely to be physiologically significant, and they make predictions about ligand-induced conformation changes i...

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Protonophoric Activity of NADH Coenzyme Q Reductase and ATP Synthase in Coupled Submitochondrial Particles from Horse Platelets

Baracca¹,

Bucchi²,

Ghelli³

et al. 1997

Biochemical and Biophysical Research Communications

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Kinetic characterization of the ATP-dependent proton pump in bacterial photosynthetic membranes: a study with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine

Cited by 13 publications

References 34 publications

Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by Δ_H+

Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by Δ_H+

Allosteric Regulation of Proton Translocation by a Vacuolar Adenosinetriphosphatase

Protonophoric Activity of NADH Coenzyme Q Reductase and ATP Synthase in Coupled Submitochondrial Particles from Horse Platelets

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Kinetic characterization of the ATP-dependent proton pump in bacterial photosynthetic membranes: a study with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine

Cited by 13 publications

References 34 publications

Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by ΔH+

Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by ΔH+

Allosteric Regulation of Proton Translocation by a Vacuolar Adenosinetriphosphatase

Protonophoric Activity of NADH Coenzyme Q Reductase and ATP Synthase in Coupled Submitochondrial Particles from Horse Platelets

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Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by Δ_H+

Sulfite Stimulates the ATP Hydrolysis Activity of but not Proton Translocation by the ATP Synthase of Rhodobacter Capsulatus and Interferes with its Activation by Δ_H+