On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis.

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.     

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.     

Demonstration and quantitation of catalytic and noncatalytic bound ATP in submitochondrial particles during oxidative phosphorylation.

Techniques are described for studying the labeling of ADP and ATP bound to the ATP synthase complex of beef heart submitochondrial particles catalyzing oxidative phosphorylation. These suffice for measurements of bound nucleotides during the time required for a single turnover, during steady state net ATP synthesis, or under quasiequilibrium conditions of ATP formation and hydrolysis. Results show that the "tightly bound" ATP associated with isolated submitochondrial particles does not become labeled by medium [32P]Pi rapidly enough to qualify as an intermediate in ATP synthesis. In contrast to chloroplast preparations, little or no bound [32P]Pi committed to ATP formation is present on particles during steady state synthesis. Also, highly active particles synthesizing ATP from [32P]Pi and filtered after EDTA addition have no detectable bound [32P]ATP even though several ATPs have been made per synthase complex. However, under quasiequilibrium conditions membrane-bound ADP and ATP are present whose labeling characteristics qualify them as intermediates in ATP synthesis. In addition, a hexokinase-accessibility approach shows the presence of a steady level of bound ATP. Lack of detection of bound intermediates under other conditions is regarded as reflecting the ready reversibility of oxidative phosphorylation, with consequent facile cleavage of bound ATP and release of bound Pi.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo.F1-ATP synthase

Fo.F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP.Mg2+ -induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319-12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 x 10(-6) M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo.F1-ATP synthase.     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.     

Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al-Shawi, M.K. & Senior, A.E. (1992), Biochemistry 31, 886-891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1 [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni-site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni-site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1.ADP + Pi <--> F1.ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1.ATP. The DeltaG degrees of the reaction with 0.54 microM F1, 2 mM Pi and 10 mM Mg2+ at pH 6.8 was -2.7 kcal.mol-1 in favor of F1.ATP. The DeltaG degrees of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, DeltaG degrees became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.     

Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP in equilibrium with 32Pi exchange catalyzed by plasma membrane Ca(2+)-ATPase from kidney proximal tubules.

The Ca(2+)-stimulated adenosine 5'-triphosphate-orthophosphate (ATP in equilibrium with 32Pi) exchange reaction was studied using a vesicular preparation derived from plasma membrane of kidney proximal tubules. With native inside-out vesicles, ATP in equilibrium with 32Pi was stimulated by micromolar Ca2+ concentrations. Treatment of the vesicles with the Ca2+ ionophore A23187 that abolished Ca2+ accumulation, strongly inhibited ATP in equilibrium with 32Pi. When Ca(2+)-ATPase was solubilized with the nonionic detergent octaethylene glycol mono n-dodecyl ether, maximal activation of ATP in equilibrium with 32Pi required millimolar Ca2+ concentrations. These Ca2+ concentrations inhibited ATP hydrolysis. ATP in equilibrium with 32Pi exhibited a Michaelian dependence on Pi and Mg2+, was stimulated by ATP, and depended on the ATP/ADP ratio. ATP in equilibrium with 32Pi was modified by the osmolytes urea, trimethylamine-N-oxide, and sucrose, which are representative of the methylamines and polyols that normally accumulate in renal tissue. These compounds did not modify the apparent affinity for Pi; they affected the response to ADP in the same fashion as the overall rate of ATP in equilibrium 32Pi, and their effects depended on medium pH. These data show that the Ca(2+)-ATPase from plasma membrane kidney proximal tubules can operate simultaneously in forward and backward directions. They also show that ATP in equilibrium with 32Pi is modulated by the ligands Ca2+, ATP, ADP, Pi, Mg2+, and H+, and by organic solutes found in renal tissue.     

Assessment of the rate of bound substrate interconversion and of ATP acceleration of product release during catalysis by mitochondrial adenosine triphosphatase

The oxygen exchange parameters for the hydrolysis of ATP by the F1-ATPase have been determined over a 140,000-fold range of ATP concentrations and a 5,000-fold range of reaction velocity. The average number of water oxygens incorporated into each Pi product ranges from a limit of about 1.02 at saturating ATP concentrations to a limit of about 3.97 at very low ATP concentrations. The latter value represents 400 reversals of hydrolysis of bound ATP prior to Pi dissociation. In accord with the binding change mechanism, this means that ATP binding at one catalytic site increases the off constant of Pi and ADP from another catalytic site by at least 20,000-fold, equivalent to the use of 6 kcal mol-1 of ATP binding energy to promote product release. The estimated rate of reversal of hydrolysis of F1-ATPase-bound ATP to bound ADP + Pi varies only about 5-fold with ATP concentration. The rate is similar that observed previously for reversal of bound ATP hydrolysis or synthesis with the membrane-bound enzyme and is greater than the rate of net ATP formation during oxidative phosphorylation. This adds to evidence that energy input or membrane components are not required for bound ATP synthesis.     

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. We have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (Pi) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]Pi, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. We also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of Pi formation is followed by a much lower, constant steady-state rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial ATP synthase: fifteen years later

Mitochondrial Fo.F1-H+-ATP synthase is the main enzyme responsible for the formation of ATP in aerobic cells. An alternating binding change mechanism is now generally accepted for the operation of the enzyme. This mechanism apparently leaves no room for the participation of nucleotides and Pi other than sequential binding to (release from) the catalytic sites. However, the kinetics of ATP hydrolysis by mitochondrial ATPase is very complex, and it is difficult to explain it in terms of the alternating binding change mechanism only. Fo.F1 catalyzes both delta muH+-dependent ATP synthesis and ATP-dependent delta muH+ generation. It is generally believed that this enzyme operates as the smallest molecular electromechanochemical reversible machine. This essay summarizes data which contradict this simple reversible mechanism and discusses a hypothesis in which different pathways are followed for ATP hydrolysis and ATP synthesis. A model for a reversible switch mechanism between ATP hydrolase and ATP synthase states of Fo. F1 is proposed.     

Specific dicyclohexylcarbodiimide inhibition of the E-P + H2O equilibrium E + Pi reaction and ATP equilibrium Pi exchange in sarcoplasmic reticulum adenosinetriphosphatase

Treatment of sarcoplasmic reticulum adenosinetriphosphatase (ATPase) with N,N'-dicyclohexylcarbodiimide is known to produce total inhibition of calcium binding and enzyme activity. However, we now find that treatment with lower reagent:protein ratios produces selective inhibition of hydrolytic Pi cleavage, enzyme phosphorylation with Pi, and ATP in equilibrium Pi exchange, while calcium binding and enzyme phosphorylation with ATP remain largely unaffected. This specific inhibition is attributed to derivatization of residues which are normally involved in acid-base-assisted catalysis of the hydrolytic reaction and its reversal, but are not involved in calcium binding or in the mechanism of phosphoryl transfer from ATP to the enzyme. This specific inhibition is prevented by the presence of micromolar calcium during the incubation with the inhibitor, evidently through an allosteric effect of calcium binding on the catalytic site. We also find that the initial adducts formed between ATPase residues and N,N'-dicyclo[14C]carbodiimide undergo further degradation with release of radioactive product into the medium, while the protein residues remain inactivated probably by linkage with neighboring residues. Therefore, the stoichiometry of radioactive labeling underestimates the actual number of inactivated residues.     

Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ～ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ～ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.     

Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.     

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.     

The noncatalytic site-deficient alpha3beta3gamma subcomplex and FoF1-ATP synthase can continuously catalyse ATP hydrolysis when Pi is present.

We investigated ATP hydrolysis by a mutant (DeltaNC) alpha3beta3gamma subcomplex of F0F1-ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites. This mutant subcomplex was activated by inorganic phosphate ions (Pi) and did not show continuous ATP hydrolysis activity in the absence of Pi. Pi also activated the wild-type alpha3beta3gamma subcomplex in a similar manner. Sulphate activated wild-type alpha3beta3gamma but not DeltaNC alpha3beta3gamma, indicating that Pi activation did not involve noncatalytic sites but that sulphate activation did. Pi also activated ATP hydrolysis and coupled proton translocation by the wild-type and DeltaNC F0F1-ATP synthases reconstituted into vesicle membranes.     

Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis.

The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled [18O]Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium [32P]ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix. The rate of bound ATP formation from bound Pi at catalytic sites was over twice the rate of the overall reaction in both states 4 and 3. The rate of reaction at the catalytic site is considerably less sensitive to the decrease in membrane potential and the concentration of medium ADP than is the rate of medium ATP formation. This supports the view that the active catalytic site is occluded and proceeds at a rapid rate which is relatively independent of delta psi m and of media substrates.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.