Bound ATP in chloroplast membranes: Formation and effect of different inhibitors on the labelling

Pflugshaupt, Christian; Bachofen, Reinhard

doi:10.1007/bf01558426

Cited by 14 publications

(1 citation statement)

References 19 publications

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“…3). However, the time course of this reaction and its sensitivity to external ATP indicate that phloridzin does not inhibit the release of ATP from a catalytic site (see also [24]). These considerations lead us to conclude that the direct precursor of the rapidly labeled tightly bound ATP is not ATP bound at the catalytic site.…”

Section: Discussionmentioning

confidence: 99%

Source of Rapidly Labeled ATP Tightly Bound to Non‐catalytic Sites on the Chloroplast ATP Synthetase

Aflalo¹,

Shavit²

1982

European Journal of Biochemistry

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1. The rates of formation and discharge of the rapidly labeled tighly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation.2. We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation.3. Since the rapidly labeled tightly bound [32P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non-catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.With the discovery of ATP and ADP tightly bound to the ATP synthetase of mitochondria and chloroplasts, the question of their participation in the mechanism of the energytransduction process has been intensively investigated [I -41. Studies on binding, dissociation and phosphorylation of nucleotides associated with the tight binding site(s) of the chloroplast ATP synthetase [5 -71 have shown that these processes are energy dependent. The different kinetic properties of the exchange (i.e. release and binding) [8] and phosphorylation [9,10] of tightly bound ADP as compared to the phosphorylation of medium ADP indicate that the deenergized conformation of the ATP synthetase contains ADP bound to a site which is not the catalytic site for ATP formation. ATP has also been detected tightly bound to chloroplast thylakoids during [lo] and after energization [6,1 I]. Measurement of the rates of exchange of phosphate oxygens during net synthesis or hydrolysis of ATP and assessment of the amounts of reactants bound during catalysis led Boyer and coworkers [6,12,13] to formulate a mechanism for energy transduction in chloroplasts and mitochondria in which ATP, tightly bound to the ATP synthetase, participates as a catalytic intermediate in oxidative and photophosphorylation. In this mechanism, the release of ATP from one catalytic site is promoted by an energy-dependent conformational change in the ATP synthetase concomitant with the binding of ADP and Pi at an alternate catalytic site.The participation of a tightly bound ATP as a necessary catalytic intermediate is questionable since during steadystate phosphorylation of various nucleoside diphosphates Pi. From the rates of formation and release of the rapidly labeled tightly bound ATP and its exchangeability with medium ATP, we deduce that this ATP species is probably bound to non-catalytic sites on the ATP synthetase. MATERIALS AND METHODSChloroplast thylakoids were isolated from fresh market lettuce leaves by conventional procedures, washed once by resuspension in 0.4 M sucrose in 1 mM Tri...

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Section: Discussionmentioning

confidence: 99%