Tentoxin has at least two binding sites on CF₁ and ϵ‐depleted CF₁ ATPases isolated from spinach chloroplast

Abstract

A new procedure for synthesis of 14C-labeled tentoxin recovery of the ATPase activity [10]. Interestingly, a tightly [14C-MePhe[(Z)A]3-tentoxin], with a high specific activity, is coupled proton transport is preserved in the partially reactidescribed. Binding experiments with CFt or CFt-E isolated from vated CF0-F1 by high concentrations of tentoxin [10], and the spinach chloroplast have been carried out using equilibrium interaction between the nucleotide binding sites, which plays a dialysis technique. The results show the presence of two classes of important role in the functioning of this complex, is severely binding sites. The association constants of the two major binding altered [11,12]. sites were derived from non-linear fitting of the binding curves.The precise binding site for tentoxin on CF1 has not been At 4°C, the first binding site has a value of K,I = 8.2 × 105 M -I yet identified. There is some evidence that tentoxin binds to in CF1 and 8.7 × 105 M -1 in CF1-E, while the second binding site the [3-subunit [3,11] although amino acids involved in the has lower affinity with K,2 = 1.5 x 104 M -1 in CF1 and 2.3 x 103 binding are still a matter of controversy [5,13]. A binding M -t in CFI-a.site located on the a-subunit or at an interface between the Key words." H+-ATPase; Chloroplast ATP synthase; subunits c~ and [3 is also a possibility [7,14]. In addition, the 14C-Labelled tentoxin; Cyclic tetrapeptide; Equilibrium binding of the 7-subunit to the ¢xl3-complex is necessary for dialysis; Spinacia oleracea L. sized by new methods, we present here an investigation of H+-ATPase. This enzyme couples the downhill proton flow tentoxin binding on spinach CF1 and CFI-~, which reveals generated by the photosynthetic redox chain with ATP synat least two types of binding sites with different affinities. thesis. Numerous studies have shown that tentoxin tightly binds to the soluble F1 moiety of the enzyme [2,4], but very little is 2. Materials and methods known about the mode of action of the toxin. The extrinsic part of chloroplastic ATP synthase, CF1, is a multi-subunit 14C-Labelled tentoxinenzyme (stoichiometry c~3133T6a) with latent ATPase activity.BocMeAla-Leu-AZPhe-GlyOMe (1) was obtained in homogeneous Removal of the E-subunit and/or chemical reduction of the phase using the synthesis scheme recently described [16] skipping the lone disulfide bond of the enzyme located on the 7-subunit methylation step of the dehydrophenylalanyl residue (AZPhe). HPLC activates ATPase activity [5]. For sensitive plants, tentoxin Rt= 23.2 min on C18 Protein&Peptide VYDAC analytical column (MeOH/H20 60/40 v/v, 0.6 ml rain-l). Using [14C]methyl iodide binding inhibits the CF1-ATPase activity at low concentration freshly synthesized [17] from [14C]methanol produced from 14CO2 (10 -8 M) [2,4,6,7] and stimulates it at a concentration of 10 -5 (specific activity 55 mCi/mmol) [18,19], selective radioactive methyla-M and higher [6][7][8][9]. The activation phenomenon also exists in tion was then performed according to [...

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…ceptible CF 1 is considered to contain a high-affinity inhibitory binding site and one or two low-affinity stimulatory binding sites (69,265,317,350). The binding of tentoxin to a lowaffinity binding site releases the inhibitory effect caused by binding of tentoxin to the high-affinity binding site and reactivates the enzyme.…”

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

“…The Two TTX Binding Sites Are Related to Inhibition and Reactivation of CF 1 -ATPase Activity-In a preceding study (27), we detected the presence of two TTX binding sites on isolated CF 1 and CF 1 -⑀, using synthetic 14 C-labeled TTX, and we related these two sites to the TTX properties of inhibition of the CF 1 -ATPase activity below 0.1 M and reactivation above 5-10 M. The present study, using TTX synthetic analogues, fully confirms the existence of these two independent TTX binding sites and their direct connection to the inhibition and stimulation processes. More, the binding parameters have been determined carefully using the two-sites formalism.…”

“…It has been demonstrated (27) that CF 1 binds two molecules of TTX on two sites of different affinities, which could be related to the inhibitory and reactivating effects of this molecule. These binding sites have not yet been identified, and the reasons for the specificity of TTX for the CF 1 -ATP synthase of some higher plants remain obscure (23,28).…”

Kinetic Analysis of Tentoxin Binding to Chloroplast F1-ATPase

“…It has been reported that CF 1 binds two molecules of TTX on distinct sites (23) presumably located on ␤-subunit, although this point remains obscure (24 -26). Since the affinity of TTX for these two binding sites was found different, we suggested that they were respectively related to the inhibitory and stimulatory activities of TTX (23). Also, Mochimaru and Sakurai (27) have recently suggested the existence of a third very low affinity binding site, which would account for the reactivation process.…”