ReveRsible pH-dependent activation / inactivation of cf 1-atpase of spinacH cHloRoplasts

the aim of the work was to study the reverse ph-dependent regulation of the enzymatic activity of the catalytic part of AtP synthase (ec 3.6.3.14) of chloroplast – coupling factor cF1. It was shown that the short-term incubation of isolated cF1 in the media with ph 4.5 or 3.5 leads to inactivation of ca 2+-AtPase, which is rapidly (t1/2 ~ 1 min) restored in the medium containing 0.5-10 mm bicarbonate at ph 7.8. After acid treatment, the rate of mg2+-ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С). The increase in Ca2+and mg2+-АТР activity of CF1 associated with the addition of Nahco3 solution was completely eliminated after the introduction of 50 mM acetazolamide – a specific inhibitor of carbonic anhydrase. the obtained results suggest the existence of the bound bicarbonate in the cF1 structure, which apparently participates in proton transfer.


ReveRsible pH-dependent activation/inactivation of cf 1 -atpase of spinacH cHloRoplasts
A. P. KhomochKIN the aim of the work was to study the reverse ph-dependent regulation of the enzymatic activity of the catalytic part of AtP synthase (ec 3. 6.3.14) of chloroplast -coupling factor cF 1 .It was shown that the short-term incubation of isolated cF 1 in the media with ph 4.5 or 3.5 leads to inactivation of ca 2+ -AtPase, which is rapidly (t 1/2 ~ 1 min) restored in the medium containing 0.5-10 mm bicarbonate at ph 7.8.After acid treatment, the rate of mg 2+ -ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С).The increase in Ca 2+ -and mg 2+ -АТР activity of CF 1 associated with the addition of Nahco 3 solution was completely eliminated after the introduction of 50 mM acetazolamide -a specific inhibitor of carbonic anhydrase.the obtained results suggest the existence of the bound bicarbonate in the cF 1 structure, which apparently participates in proton transfer.K e y w o r d s: spinach chloroplasts, cF 1 -AtPase, activation/inactivation, AtP hydrolysis, ph-dependence, acetazolamide.
ATP synthase (EC 3.6.3.14) is a membrane enzymatic complex which synthesis and hydrolysis of ATP coupled with the transmembrane proton transfer in chloroplasts, mitochondria and bacteria.It consists of a hydrophobic part F 0 , which functions as a proton channel and a hydrophilic part -a coupling factor F 1 , which contains several nucleotide binding sites and performs catalytic function [1,2].Proton ATP synthases from various organisms have similar structure.This is largely characteristic of chloroplast ATPase catalytic part -the CF 1 factor, which is water-soluble enzyme and consists of five types of polypeptides in stoichiometric ratio α:β:γ:δ:ε ~ 3:3:1:1:1 [2][3][4].The central position of the ATP synthase complex in the energy supply of a living cell is determined by the need of precise regulation of its functioning and coordinated with the physiological state of an organism and its demand for energy.To date, several mechanisms have been identified that are involved in the regulation of ATP synthase.After separation from the membrane, the catalytic part of the complex, the isolated coupling factor CF 1 , loses the ability to catalyse the ATP synthesis, but retains ATPase activity.In this case, the isolated CF 1 is a latent (hidden) ATPase and catalyses the hydroly sis of ATP only after activation by heat [5] or as a result of treatment with redox reagents, alcohols and some detergents [2].A significant activation of ATP hydrolysis is also achieved when adding oxyanionbicarbonate, sulphite, phosphate, etc., to the reaction medium.[2,5,6].
The enzymatic activity of membrane-bound ATP synthases is primarily controlled by the value of the transmembrane proton gradient and realized during proton transfer through polypeptides of the complex during protonation/ deprotonation of certain amino acid groups [6,7].The protonation of specific groups in the isolated CF 1 factor can simulate the energization process.Analysis of the pHdependence of Mg 2+ -ATPase reaction catalyzed by the isolated factor CF 1 made it possible to estimate the dissociation constant of functionally important residues (pK 5.8-6.7)[6], herewith it was shown that the kinetic behavior of CF 1 -ATPase is controlled by the degree of protonation of these groups.According to the data of Malyan [8], the activity of Mg 2+ -ATPase increases significantly after incubation of the isolated coupling factor CF 1 at pH 5.5 and is in-hibited again when CF 1 is transferred to a weakly alkaline medium that indicates the existence of at least two functional states of the coupling factor, reversible transitions between them occurring with pH changes.
The aim of this work was to study the pH-dependent inactivation/reactivation of Са 2+ -and Mg 2+ -ATPase activity of the isolated coupling factor CF 1 of spinach chloroplasts and the effect of specific inhibitor of carbonic anhydrase -acetazolamide on these processes.

Materials and Methods
Chloroplasts were isolated from fresh spinach (Spinacia oleracea L.) leaves as described previously in [9] and disrupted for 10 min in hypotonic medium that contained 5 mM Tris-НСl (pH 7.8) and 10 mM NaCl.The thylakoids were washed twice with a hypotonic solution, reprecipitated for 10 min at 15,000 g and used to isolate the preparation of the coupling factor CF 1 by the method of [10] with some modifications.All isolation operations of thylakoids and CF 1 were performed at 4 °C.The chlorophyll concentration in the preparations of the thylakoid membranes was determined according to Arnon [11], the protein concentration -according to Lowry [12].The purity of the obtained CF 1 preparation was assessed by the results of electrophoresis with charge shift as described in [13].
The latent isolated CF 1 -ATPase was activated by heating; to do this the preparation (1.5-2.0 mg) was added to 1 ml of a solution containing 10 mM ATP, 25 mM Tris-HCl (pH 7.9), and 10 mM CaCl 2 (or MgCl 2 ).The mixture was placed on a 37 °C water bath and incubated for 30 min, after that it was transferred to a container with water (26 °C).ATPase reaction was stopped by adding 1 ml of 8% trichloroacetic acid (TCA) solution to the mixture.ATPase activity was determined by measu ring concentrations of released inorganic phosphorus (P i ) in the reaction medium that contained 15 mM Tris-HCl, pH 7.9, 5 mM ATP and 5 mM CaCl 2 (or MgCl 2 ) (at 26 °C or 37 °C) and expressed as μmol The amount of P i in the sample was determined by the Lowry and Lopez method in the Skulachev modification [14].
To determine the dependence of ATPase activity on pH, the CF 1 solution was incubated for 5 min in the media which contained 10 mM succinate and 50 mM Tris-HCl with pH of 3.5; 4.5; 5.4 and 7.8.After a 5-minute incubation, CF 1 -ATPase was activated by heating and the rate of Ca 2+ or Mg 2+ -dependent ATP hydrolysis was determined.
For ATPase reactivation after 5-minute acid incubation at pH 3.5; 4.5 or 5.4, the calculated amount of 1 M NaOH solution was added to the medium so that the final pH of the solution was 7.8.ATPase activity of the enzyme in this series of experiments was determined after 10 min incubation at pH 7.8.
All studies were performed in triplicate.Student's t-test was used to compare the samples, the differences were considered reliable at P <0.05.The figures and the table show the mean values and their standard errors.

Results and discussion
Table 1 shows the results of determination of Ca 2+ -and Mg 2+ -dependent ATPase activity after 5 min of incubation of isolated CF 1 at different pH.It is evident that Ca 2+ -ATPase CF 1 , previously incubated under physiological pH (≈ 7.8), increased significantly during the 30-minute exposure at 37 °C.
At all pH values, the Ca 2+ -dependent ATPase activity at 26 °C was noticeably lower than the measurements made at 37 °C, that agrees with [2,5].It should also be noted that Ca 2+ -ATPase activity of the isolated coupling factor CF 1 after preliminary incubation at low pH values (3.5, 4.5 or 5.4) was significantly inhibited compared to the control and at pH 7.8 both at 26 °C and at 37 °C.
In contrast to Ca 2+ -ATPase, Mg 2+ -ATPase activity was practically unchanged during the 30-minute incubation of the preparations at 37 °C, preincubated at reduced pH (Table 1).At the same time, there was a more than triple increase in the rate of Mg 2+ -dependent ATP hydrolysis in samples incubated at pH 3.5 in comparison with the control (pH 7.8).However it should be noted that the level of Mg 2+ -ATPase activity remained low for all pH and temperatures studied.
The question of whether the inactivation of CF 1 -ATPase at low pH was irreversible was solved in the following series of experiments (Table 2).First, the solution of desalted isolated coupling factor CF 1 t a b l e 1. ca 2+  was incubated at pH 3.5 for 5 min, then the solution pH was increased to 7.8.In the variants indicated (Table 2) 1 mM NaHCO 3 or 50 μM of specific carbonic anhydrase inhibitor acetazolamide (AZ) was added to the mixture.The data of Table 2 show, that Ca 2+ -ATP activity of the preparation pre-treated at pH 3.5 increases after the subsequent 10 min incubation in the reaction solution with pH 7.8, exceeding almost twice the activi ty of Ca 2+ -ATPase determined immediately after the acidic incubation.This indicates the reversibility of the acid inactivation of the enzyme and the possibility of its reactivation in the case of transfer to a slightly alkaline environment.A similar effect was previously described by Malyan [6,8] in the study of the pH dependence of the Mg 2+ -ATPase activity of isolated CF 1 .
In our experiments, the rate of ATP hydrolysis increased significantly, when 1 mM NaHCO 3 was added to the medium (pH 7.8) in the final incubation (Table 2).The addition of 50 μM acetazolamide, a specific inhibitor of carbonic anhydrase, to the reaction mixture had practically no effect on the rate of ATP hydrolysis but eliminated the stimulating effect of bicarbonate on Ca 2+ -and Mg 2+ -ATPase.Previous-

T a b l e 2. The effect of bicarbonate and acetazolamide (AZ) on the reactivation of Са 2+ -and mg 2+ -AtPase activity of the isolated factor cF 1 (m ± m, n = 3)
Additives during incubation, 30 min, pH 7.8, 37 °С Са 2+ -ATPase activity, μmol•(mg protein) - Note: pH of initial incubation was 3.5 ly, we observed [4] the elimination of bicarbonate stimulation of photosynthetic phosphorylation in isolated thylakoids after addition of acetazolamide.
The results of Table 2 indicate a similarity in the character of the reactivation of Ca 2+ -and Mg 2+ -ATPases after the acid treatment of CF 1 .The activity in both cases increased with incubation in a slightly alkaline medium (pH 7.8), and when the bicarbonate anion was added, Mg 2+ -ATPase was stimu lated almost 4 times, and Ca 2+ -ATPase -2 times.The effective concentration of the anion (1 mM) was much lower than the published value for activation of Mg 2+ -ATPase [15,16].In our experiments the sensitivity to the bicarbonate anion was increased for factor CF 1 treated at low pH, and the difference between Mg 2+ -dependent and Ca 2+ -dependent ATPase activities decreased (Table 2).
It can be seen that the rate of ATP hydrolysis depended on temperature.Са 2+ -ATPase activity was stimulated with bicarbonate both at 26 °C and at 37 °C (Fig. 1).Addition of 0.1-1 mM of NaHCO 3 led Fig. 1.Са 2+ -АТРase activity dependence on NaHCO 3 concentration at рН 7.8 after acid inactivation of cF 1 -АТРase Fig. 2. mg 2+ -АТРase activity dependence on Nahco 3 concentration at рН 7.8 after acid inactivation of cF 1 -АТРase to a sharp increase in the ATP hydrolysis rate, and maximum stimulation was observed at 37 °C with bicarbonate in concentrations exceeding 1 mM.Mg 2+ -ATPase activity was stimulated with bicarbonate only at 37 °C, whereas at 26 °C bicar- bonate did not affect the rate of ATPase reaction (Fig. 2).
The dynamics of the development of Са 2+ -and Mg 2+ -ATPase activity at 37 °C in the presence of 1 mM NaHCO 3 in the medium is shown in Fig. 3.
Samples of isolated CF 1 after a 5-minute incubation at pH 3.5 were transferred to the medium with pH 7.8, they were incubated for 30 seconds, 1, 2, 5 and 10 min and added to the reaction mixture containing 1 mM NaHCO 3 , ATPase reaction was activated by heating.It can be seen that half-maximal activation of Ca 2+ -and Mg 2+ -ATPase was achieved after approximately 1.5 and 2 min, respectively.According to Malyan [6], the reactivation of CF 1 after incubation with pH of 5.5 was much slower and required 11 min to achieve half-maximal activity.
The results obtained in the present work show that the enzymatic activity of the catalytic part of the ATP synthase of chloroplasts -factor CF 1 in the reaction of ATP hydrolysis is inhibited after its incubation in a medium with pH <5.5.The inhibition is reversible and partially eliminated, when the enzyme is transferred to a medium with pH of 7.8.If bicarbonate is present in the solution, the reactivation of ATPase to the control level of enzymatic activity occurs much faster.
Bicarbonate is a known stimulator of Mg 2+ -ATPase activity of CF 1 [6,15,16].However, but ac- cording to the cited studies, its effective concentration is more than an order of magnitude higher than bicarbonate concentration, which was effective in the reactivation of CF 1 -ATPase after acid treatment in our work (Fig. 2 and 3).Previously, we reported the presence of carbonic anhydrase activity in isolated coupling factor CF 1 [13,19].The results of this study suggest that CF 1 -carbonic anhydrase is involved in the process of reactivation of ATPase activity after acid inhibition.This is evidenced by data on the negative effect of acetazolamide, the inhibitor of carbonic anhydrases, on the process of reactivation of the ATPase reaction (Table 2).