[Effects of Medium Viscosity Increasing Agents on ATP Synthesis in Chloroplast Thylakoids].

The effect of an increase in the medium viscosity on cyclic photophosphorylation in chloroplast thylakoids and on Ca2+ -dependent ATP hydrolysis by the chloroplast coupling factor CF, was studied. With 0.1-0.2 mM ADP used it was found that the rate of ATP synthesis decreases after addition of various agents that increase the medium viscosity (sucrose, dextran 40 or polyethylene glycol 6000 provided that these agents cause neither uncoupling nor electron transport inhibition in the absence of ADP. Dextran and polyethylene glycol inhibited ATP synthesis by 50% when their concentrations were much lower (6-10%) than that of sucrose (30-40%), while 50% inhibition of Ca2+ -dependent ATP hydrolysis by CFI-ATPase was observed at higher concentrations of dextran and polyethylene glycol (9-13%) and lower concentrations of sucrose (about 20%). For ADP, the effective Michaelis constant (KM) was shown to increase 2-3-fold with the increasing viscosity; meanwhile the maximal rate of cyclic photophosphorylation remained virtually unchanged. The dependence of K(M) on the medium viscosity can serve as a criterion for the process of diffusion-controlled photophosphorylation. Possible mechanisms of ADP and ATP diffusion are discussed.

Noncatalytic nucleotide binding sites: properties and mechanism of involvement in ATP synthase activity regulation.

ATP synthases (FoF1-ATPases) of chloroplasts, mitochondria, and bacteria catalyze ATP synthesis or hydrolysis coupled with the transmembrane transfer of protons or sodium ions. Their activity is regulated through their reversible inactivation resulting from a decreased transmembrane potential difference. The inactivation is believed to conserve ATP previously synthesized under conditions of sufficient energy supply against unproductive hydrolysis. This review is focused on the mechanism of nucleotide-dependent regulation of the ATP synthase activity where the so-called noncatalytic nucleotide binding sites are involved. Properties of these sites varying upon free enzyme transition to its membrane-bound form, their dependence on membrane energization, and putative mechanisms of noncatalytic site-mediated regulation of the ATP synthase activity are discussed.

Role of short conserved segments of alpha- and beta-subunits that link F(1)-ATPase catalytic and noncatalytic sites.

An analysis of amino acid sequences and 3D structures of chloroplast, mitochondrial, and bacterial F(1)-ATPases revealed that in their alpha- and beta-chains there are short highly conserved segments linking in pairs the catalytic and noncatalytic sites. The analysis was based on the reported effect of directed mutagenesis of amino acids forming these segments on catalytic properties of the F(1)-ATPases. It is proposed that one of these segments is responsible for transduction of a conformation signal from the noncatalytic to catalytic site upon ADP-for-ATP substitution at the noncatalytic site. At the catalytic site, this signal changes position of the terminal amino acid residue with respect to the adenine part of the molecule and results in a lower tightness of MgADP binding and its dissociation followed by enzyme activation. Mutagenesis of amino acids comprised by the two other segments was shown to produce an effect on the rate of cooperative catalysis, whereas the rate of single-site catalysis remained unaffected. This suggests that these segments are responsible for the cooperative mode of enzyme functioning.

Interaction of pyrophosphate with catalytic and noncatalytic sites of chloroplast ATP synthase.

The effect of pyrophosphate (PP(i)) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PP(i) competed with nucleotides for binding to noncatalytic sites. In the dark, PP(i) was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their subsequent interaction with ADP and ATP. The effect of PP(i) on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PP(i) inhibited ATPase activity of ATP synthase. Addition of PP(i) to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated its activity during prolonged incubation. These results indicate that the stimulating effect of PP(i) light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites. The inhibition of ATP synthase results from competition between PP(i) and ATP for binding to catalytic sites.

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF1.

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.

Light-dependent incorporation of adenine nucleotide into noncatalytic sites of chloroplast ATP synthase.

The binding of ADP and ATP to noncatalytic sites of dithiothreitol-modified chloroplast ATP synthase was studied. Selective binding of nucleotides to noncatalytic sites was provided by preliminary light incubation of thylakoid membranes with [14C]ADP followed by its dissociation from catalytic sites during dark ATP hydrolysis stimulated by bisulfite ions ("cold chase"). Incorporation of labeled nucleotides increased with increasing light intensity. Concentration-dependent equilibrium between free and bound nucleotides was achieved within 2-10 min with the following characteristic parameters: the maximal value of nucleotide incorporation was 1.5 nmol/mg of chlorophyll, and the dissociation constant was 1.5 microM. The dependence of nucleotide incorporation on Mg2+ concentration was slight and changed insignificantly upon substituting Ca2+ for Mg2+. Dissociation of nucleotide from noncatalytic sites was illumination-dependent. The dissociation kinetics suggested the existence of at least two nucleotide-binding sites with different dissociation rate constants.

ATP binding to noncatalytic sites of chloroplast coupling factor CF1.

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF1 was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF1 gamma-subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF1 with Mg2+ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant kd(ATP) approximately/= 10-3 min-1. Noncatalytic sites of CF1 were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF1 with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.

Interaction of sulfite with the noncatalytic and catalytic sites of chloroplast coupling factor cf1.

The interaction between sulfite, an efficient Mg2+-dependent F1-ATPase activator, and chloroplast CF1-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF1. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF1 catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg2+-dependent ATPase activity of CF1 is caused by sulfite binding to its catalytic sites.

Study of the structure of the thylakoid membrane-bound chloroplast coupling factor CF1.

The structure of thylakoid membrane-bound chloroplast coupling factor CF1 was studied by limited proteolysis followed by sodium dodecylsulfate polyacrylamide gel electrophoresis and N-terminal sequence analysis. The N-terminal fragment of the alpha-subunit was shown to have an exposed area including the peptide bond R21-E22. The cleavage of this peptide bond caused the alphaK24-V25 bond to be exposed to the outside. In the N-terminal fragment of the beta-subunit, the L14-E15 bond was identified and found to be subject to trypsinolysis. Also, the alphaR140-S141, alphaG160-R161, and betaG102-G103 bonds were accessible to the proteolytic attack. In general, the beta-subunit of membrane-bound CF1 is more sensitive to proteolysis than that of solubilized CF1. The products of proteolysis of the alpha-subunit did not contain the polypeptides typical of the reaction of cleavage of the alphaE17-G18 and alphaE22-V23 bonds in isolated CF1. These results suggest a significant structural difference between soluble and membrane-bound CF1. A number of peptide bonds, alphaG160-R161 in particular, were shown to be shielded from proteolytic attack by papain in illuminated thylakoid membranes, probably as a result of membrane energization. In contrast, the light-induced reduction of the gamma-subunit caused an increase in the accessibility of some peptide bonds to this protease, including the alphaG160-R161 bond.

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase.

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [(14)C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [(14)C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m(-2), ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.

Catalytic properties of ß subunit isolated from chloroplast coupling factor 1.

The chloroplast coupling factor (CF1) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the ß subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The ß subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg(2+)-dependent ATPase activity of CF1. However, it manifested appreciable adenylate kinase-like and ATP-ADP γ-phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg(2+) ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg(2+)-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF1-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the ß subunit.

pH dependent changes in ADP and ATP affinity for the tight nucleotide-binding site of chloroplast coupling factor 1.

The pH-dependence of ADP and ATP affinity for CF1 tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF1 ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF1 ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF1 tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.