Application of HPLC to the Purification of Coupling Factor CF<sub>1</sub>From Spinach Chloroplasts and of Some of its Subunits

Berger, G.; Girault, G.; Andre, F; Galmiche, J. M.

doi:10.1080/01483918708066783

Cited by 35 publications

(10 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Storage conditions and determination of the concentration of CF 1 -⑀ were described previously (31). The assays were modified slightly with respect to previous conditions (30).…”

Section: Methodsmentioning

confidence: 99%

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Santolini¹,

Haraux²,

Sigalat³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Eight synthetic analogues of tentoxin (cyclo-(L-N-MeGlu1 -L-Leu 2 -N-Me⌬ Z Phe 3 -Gly 4 )) modified in residues 1, 2, and 3 were checked for their ability to inhibit and reactivate the ATPase activity of the activated soluble part of chloroplast ATP synthase. The data were consistent with a model involving two binding sites of different affinities for the toxins. The occupancy of the high affinity site (or tight site) gave rise to an inactive complex, whereas filling both sites (tight ؉ loose) gave rise to a complex of variable activity, dependent on the toxin analogue. Competition experiments between tentoxin and nonreactivating analogues allowed discrimination between the absence of binding and a nonproductive binding to the site of lower affinity (or loose site). The affinity for the loose site was not affected significantly by the modifications of the tentoxin molecule, whereas the affinity for the tight site was found notably changed. Increasing the size of side chain 1 or 2 and introducing a net electrical charge both resulted in a decrease of affinity for the tight site, but the second change dominated the first one. The activity of different ternary complexes enzyme-tentoxin-analogue depended on the nature of the toxin bound on each site and not only on that bound on the loose site. This demonstrates that the reactivation process results from an interaction, direct or not, between these two binding sites. Possible molecular mechanisms are discussed.F 0 F 1 proton ATPases (or ATP synthases) are bound to energy-transducing membranes and couple the phosphorylation of ADP into ATP to the dissipation of a protonmotive force. They consist of a transmembrane proton channel (F 0 ) and an extrinsic part (F 1 ) bearing six nucleotide binding sites, catalytic and noncatalytic. The F 1 moiety is composed of five different subunits named ␣, ␤, ␥, ␦, and ⑀ (stoichiometry ␣ 9 -12]), the mitochondrial enzyme having additional subunits (5, 6). It is proposed that the F 0 moiety would work as a rotative proton-driven motor, the rotor consisting of the c subunits (7), presumably arranged in a crown (8). The rotation would be transmitted to the ␥ subunit of the F 1 moiety (9), which should modify sequentially the three catalytic sites located on ␤ subunits (4) to induce ATP synthesis (10). Experimental arguments have been presented against (11,12) and for (9, 13-15) the rotation of ␥. An essential feature of this model is that the cooperative functioning among the three catalytic sites is strictly related to the rotation of the ␥ subunit and thus to the proton pumping activity.[), produced by several phytopathogenic fungi of the Alternaria genus (16,17). Under special conditions, this toxin induces a chlorosis in some higher plants (18). It specifically inhibits ATP synthesis in isolated chloroplasts (19). In vitro and at low concentrations (10TTX inhibits the isolated chloroplast F 1 -ATPase (19 -22), but at higher concentrations (10 Ϫ5 -10 Ϫ4 M), it strongly stimulates ATPase activity (21-23). At these same concentration...

show abstract

“…Storage conditions and determination of the concentration of CF 1 -⑀ were described previously (31). The assays were modified slightly with respect to previous conditions (30).…”

Section: Methodsmentioning

confidence: 99%

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Santolini¹,

Haraux²,

Sigalat³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…15 Nucleotide binding was measured according to the method of Hummel and Dreyer, adapted to ATPase: 2,16-19 a known quantity of ATPase is injected on a gel filtration column (TSK 2000 SW, 7.5 mm x 300 mm) or on an anion exchange column (TSK DEAE 2 SW, 4.6 mm x 250 mm) equilibrated with a fixed concentration of ADP, in Tris buffer 0.075 M, pH 8.5 containing variable concentrations of Mg 2+ . The ligand which is bound to the protein migrates with it and is withdrawn from the solvent.…”

Section: Methodsmentioning

confidence: 97%

USE OF HPLC FOR THE STUDY OF ADP BINDING TO CHLOROPLAST ATPase. I. INFLUENCE OF EXPERIMENTAL CONDITIONS AND PROPOSITION OF MECHANISM

Berger

Girault

Zimmermann

2000

Journal of Liquid Chromatography & Related Technologies

Self Cite

View full text Add to dashboard Cite

The binding of ADP to chloroplast ATPase (CF 1 ) has been studied by chromatographic methods (Hummel and Dreyer method and chromatographic determination of the enzymatic inhibition).The influence of different factors has been studied: temperature, ionic strength, activation treatments, pyrophosphate fixation. From the comparison of the dependence of ADP binding (and in certain cases, of ATP binding), with that of the enzymatic activity, it is shown that the conclusions drawn for the dissociation constants cannot be extended to the Michaelis constant.The role of the lysine present in the nucleotide binding sites, already emphasized by several authors, has been proposed to be the ligand, in the protonated form, of the negatively charged phosphate groups of ADP or ATP.

show abstract

“…44 The enzyme was activated by incubation for 3 h at room temperature at a concentration of 80 kg ml~1 in a medium containing Tricine (20 mM) and DDT (3 mM), pH 8É0. For assays of ATP hydrolysis, the activated enzyme was diluted 40-fold in the reaction medium containing (50 mM), Tris-SO 4 MgSO 4 (0É18 mM), (40 mM) pH 8É0.…”

Section: Tentoxin : Assays On Isolated Cfmentioning

confidence: 99%

Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxin

et al. 1998

View full text Add to dashboard Cite

: Difficulties in synthesis make natural cyclopeptides challenging targets for chemists. Our interest focused on two natural toxic cyclopeptide series produced by pathogenic fungi :The total syntheses of these two bioactive series were optimised, and several analogues were designed and synthesised to establish structureÈactivity relationships. The importance of synthetic analogues in the identiÐcation of molecular targets and the explanation of mechanisms of action was demonstrated. Such systematic investigations can determine the crucial features responsible for the activity of the natural compound and help the design of more powerful or more selective products.1998 SCI. ( Pestic. Sci., 52, 81È89, 1998 Key words : cyclopeptide ; cyclodepsipeptide ; tentoxin ; H`-ATPase ; herbi-F 0 F 1 cide ; destruxin ; insecticide Abbreviations : These follow the recommendations of the IUPAC-IUB Commission on Biological Nomenclature as given in Eur. J. Biochem. 138 (1984), 9È37.

show abstract

Application of HPLC to the Purification of Coupling Factor CF₁From Spinach Chloroplasts and of Some of its Subunits

Cited by 35 publications

References 15 publications

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

USE OF HPLC FOR THE STUDY OF ADP BINDING TO CHLOROPLAST ATPase. I. INFLUENCE OF EXPERIMENTAL CONDITIONS AND PROPOSITION OF MECHANISM

Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxin

Contact Info

Product

Resources

About

Application of HPLC to the Purification of Coupling Factor CF1From Spinach Chloroplasts and of Some of its Subunits

Cited by 35 publications

References 15 publications

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

USE OF HPLC FOR THE STUDY OF ADP BINDING TO CHLOROPLAST ATPase. I. INFLUENCE OF EXPERIMENTAL CONDITIONS AND PROPOSITION OF MECHANISM

Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxin

Contact Info

Product

Resources

About

Application of HPLC to the Purification of Coupling Factor CF₁From Spinach Chloroplasts and of Some of its Subunits