Oxidative phosphorylation in pea cotyledon submitochondrial particles

Mitochondria and submitochondrial particles (SMP) from pea cotyledons were shown to catalyze oxidative phosphorylation as measured by ³²Pi uptake into phosphate esters. ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomycin. Experiments with the adenine nucleotide translocator inhibitor atractyloside indicated the SMP were inside-out. Mersalyl completely inhibited ATP synthesis by SMP, and a separate experiment indicated that mersalyl has a direct effect on the ATPase complex. The kinetics of ATP synthesis indicated a high affinity for phosphate (K_m = 0.18 millimolar). ADP kinetics gave a biphasic curve with K_m values of about 4.8 and 160 micromolar. O₂ uptake and ATP synthesis had a pH maximum of 7.6 while the ratio of micromoles phosphate esterified to microatoms O₂ taken up was highest at pH 7.2. Sodium chloride inhibited both ATP synthesis and O₂ uptake but stimulated the ATPase reaction. The SMP also catalyzed a slow ATP-phosphate exchange reaction.     

Exploring sites on mitochondrial ATPase for catalysis,regulation, and inhibition

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.     

Interaction of aurovertin with submitochondrial particles,deficient in ATPase inhibitor

1. Binding of aurovertin to submitochondrial particles deficient in ATPase inhibitor is accompanied by an enhancement of the fluorescence by at least 100-fold.2. This change in fluorescence proceeds in three phases. The slowest change may be due to a conformational change in F₁, induced by the antibiotic bound during the rapid phases, giving rise to an increase in the quantum yield of the bound fluorochrome.3. Phosphate and ATP quench the fluorescence of the particle-aurovertin complex and ADP enhances it; the rate and extent of these changes are dependent on the availability of free Mg²⁺.4. There is at least one binding site on the submitochondrial particles, where ATP, ADP and phosphate can bind reversibly and for which these ligands compete. These interactions are dependent on the availability of free Mg²⁺ and are partly sensitive to oligomycin.5. Binding studies reveal two binding sites for aurovertin on inhibitor-free particles, one with high affinity and one with a lower affinity. Ligands such as phosphate and ATP decrease both the quantum yield and the affinity of the particles for aurovertin. They also increase the total concentration of binding sites, and affect the relative contribution of weak and strong binding sites.6. A model is presented in which changes of the aurovertin fluorescence reflect conformational changes of the ATPase induced by its ligands.     

Adenosine triphosphatase from rat liver mitochondria: separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.

Homogeneous ATPase from rat liver mitochondria binds one mole of ADP per mole of enzyme reversibly, and with high affinity (K_D = 1–2 μM). The high affinity binding site is highly specific for ADP and dADP. AMP does not bind. Agents which inhibit ATP hydrolysis have little inhibitory effect on the high affinity binding of ADP. These agents include adenylyl imidodiphosphate (AMP-PNP), azide, sucrose, and the divalent cation Mg⁺⁺. AMP-PNP inhibits ATPase activity in phosphorylating membrane preparations of rat liver mitochondria by about 90 percent, but is without effect on ATP synthesis. These results are consistent with the view that the purified soluble, and the membrane-bound ATPase of rat liver mitochondria contain separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.     

An inhibitory high affinity binding site for ADP in the oligomycin-sensitive ATPase of beef heart submitochondrial particles.

Kinetic evidence are presented for the existence of a high affinity inhibitory site for ADP /K_i < 10^?7 M/ in the oligomycin-sensitive ATPase of beef heart submitochondrial particles. The ATPase·ADP complex is completely inactive in the ATPase reaction; it can be converted into active ATPase in a slow ATP-dependent reaction. The dependence of a first order rate constant for activation of the enzyme·ADP complex on concentration of ATP gives a K_m value equal to that for ATP in the ATPase reaction. The data obtained suggest that the membrane-bound ATPase complex contains two kinetically distinct nucleotide-binding centers, i.e. center 1 binds ATP or ADP with a formation of enzyme-substrate or enzyme-competitive inhibitor complexes: center 2 binds ADP with a formation of a complex which is able to bind ATP in center 1 and unable to hydrolyze the bound ATP. The binding of ATP or ADP in center 1 changes the reactivity of center 2 towards ADP.     

Adenylate kinase is a source of ATP for tumor mitochondrial hexokinase

It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado₂P₅, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado₂P₅ does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent K_m of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, K_D = 1–2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (K_I = 240–300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, P_i can maintain the active form of the enzyme.     

Characteristics of adenylyl imidodiphosphate- and ADP-binding sites insoluble and particulate mitochondrial ATPase. Studies with methanol

The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In soluble F1-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F1-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP in ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (K_i = 9.2 × 10^?7 M) and the bound oligomycin-sensitive APTase (K_i = 1.3 × 10^?6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (K_i = 2.0 × 10^?7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.     

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O₂ uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O₂ uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (K_m 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (K_m 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.     

Mechanistic basis for differential inhibition of the F1Fo‐ATPase by aurovertin

The mitochondrial F₁F_o‐ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F₁F_o‐ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F₁ domain and inhibits F₁F_o‐ATPase‐catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F₁F_o‐ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K_i values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F₁F_o‐ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single‐molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F₁F_o‐ATPase catalyzes ATP synthesis and hydrolysis. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 830–840, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles

1. The reversible equilibrium between the mitochondrial ATPase (F₁) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions.2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities.3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP.4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation.5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor.6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles.7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F₁-aurovertin complex.8. It is suggested that the ATPase inhibitor brings F₁ in a conformation denoted 1F₁ that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.     

Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.     

Hexokinase II of Pea Seeds

A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K_m, 48 micromolar) for glucose and a relatively low affinity (K_m, 10 millimolar) for fructose. The K_m for MgATP was 86 micromolar. Mg²⁺ was required for activity, but excess Mg²⁺ was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al³⁺ was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed.     

Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria

Background

Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.

Methodology/Principal Findings

The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically) and phosphorylation (determined using the glucose - hexokinase - glucose-6-phosphate dehydrogenase - NADP⁺ enzymatic system) rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K_mVox) and phosphorylation (K_mVp) rates. We also demonstrate that determination of K_mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.

Conclusions/Significance

Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.     

Content of adenosine phosphate compounds in pea roots grown in saline media

The levels of ATP, ADP and AMP, the activity of phosphatases, and the ability for oxidative phosphorylation were studied in roots of pea (Pisum sativum) plants grown in media salinized either with NaCl or Na₂SO₄. In response to salinity, the ATP level in the roots decreased, whereas the ADP level increased slightly. As a result, the ADP:ATP ratio in the tissue increased with increasing salinity in the growth medium. The AMP level in the tissue was not affected by salinity.     

Heart FoF1-ATPase changes during the acute phase of Trypanosoma cruzi infection in rats

The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K_0.5 values, and a decreased total V_max. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100 - solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.     

ATP and ADP hydrolysis in cell membranes from rat myometrium

Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K _m values were 506.4?±?62.1 and 638.8?±?31.3?μM, with a calculated V _max (app) of 3,973.0?±?279.5 and 2,853.9?±?79.8?nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5?mM). According to similar apparent K_m values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology.