Phosphofructokinase from lycopersicon esculentum fruits-I. Kinetic properties in relation to its substrates

The kinetic properties of phosphofructokinase with regard to its substrates are discussed. Free ATP is inhibitory to the enzyme while the Mg-ATP complex at a concentration up to 5 mM is not. The kinetics with respect to Mg-ATP follow simple Michaelis-Menten kinetics and this pattern is not affected by changes in concentration of the second substrate fructose-6-phosphate (F6P). The kinetics with respect to F6P showed apparent negative co-operative interactions in the presence of saturating levels of Mg²⁺ relative to ATP. In the presence of inhibitory levels of free ATP, the kinetics showed positive co-operative interactions. The relationship between the nature of the kinetics of the enzyme with F6P and the various molecular forms of PFK are discussed.     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Effect of low pH on single skeletal muscle myosin mechanics and kinetics

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V(actin)) in the IVM by 36%. Single molecule experiments, at 1 microM ATP, decreased the average unitary step size of myosin (d) from 10 +/- 2 nm (pH 7.4) to 2 +/- 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 microM to 1 mM at pH 6.4 restored d (9 +/- 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V(actin), however, was not restored by raising the [ATP] (1-10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t(on)). Measurement of t(on) as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t(on) by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V(actin) approximately d/t(on)), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V(actin), suggesting a molecular explanation for this component of muscular fatigue.     

Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange.

The mammalian 70-kilodalton heat shock cognate protein (Hsc70) is an abundant, cytosolic molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis. In vitro, purified Hsc70 was found to have a slow, intrinsic ATPase activity in the absence of protein substrates. The addition of an unfolded protein such as apocytochrome c stimulated ATP hydrolysis 2-3-fold. In contrast, the native holoprotein, cytochrome c, did not stimulate the ATPase rate, in accord with recent observations that 70-kilodalton heat shock proteins interact selectively with unfolded proteins. Stimulation of ATP hydrolysis by apocytochrome c was due to an increase in the Vmax, with no effect on the Km for ATP. Following hydrolysis of [3H]ATP, a relatively stable [3H]ADP.Hsc70 complex was formed. Release of [3H]ADP from Hsc70 was most efficient in the presence of other nucleotides such as ADP or ATP, suggesting that ADP release occurs as an ADP/ATP exchange reaction. The loss of radiolabeled ADP from Hsc70 in the presence of exogenous nucleotides followed first-order kinetics. In the presence of nucleotides, apocytochrome c induced a 2-fold increase in the rate of ADP release from Hsc70. Moreover, rate constants of the nucleotide exchange reaction measured in the absence and presence of apocytochrome c (0.16 and 0.34 min-1, respectively) closely matched the kcat values derived from ATP hydrolysis measurements (0.15 and 0.38 min-1, respectively). The results suggest that ADP release in a rate-limiting step in the Hsc70 ATPase reaction and that unfolded proteins stimulate ATP hydrolysis by accelerating the rate of ADP/ATP exchange.     

Dimeric Eg5 maintains processivity through alternating-site catalysis with rate-limiting ATP hydrolysis

Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acid-quench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P(i) release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.     

Selective stabilization of a partially unfolded protein by a metabolite

When proteins fold in vivo, the intermediates that exist transiently on their folding pathways are exposed to the potential interactions with a plethora of metabolites within the cell. However, these potential interactions are commonly ignored. Here, we report a case in which a ubiquitous metabolite interacts selectively with a nonnative conformation of a protein and facilitates protein folding and unfolding process. From our previous proteomics study, we have discovered that Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is not known to bind ATP under native conditions, is apparently destabilized in the presence of a physiological concentration of ATP. To decipher the origin of this surprising effect, we investigated the thermodynamics and kinetics of folding and unfolding of GAPDH in the presence of ATP. Equilibrium unfolding of the protein in urea showed that a partially unfolded equilibrium intermediate accumulates in the presence of ATP. This intermediate has a quaternary structure distinct from the native protein. Also, ATP significantly accelerates the unfolding of GAPDH by selectively stabilizing a transition state that is distinct from the native state of the protein. Moreover, ATP also significantly accelerates the folding of GAPDH. These results demonstrate that ATP interacts specifically with a partially unfolded form of GAPDH and affects the kinetics of folding and unfolding of this protein. This unusual effect of ATP on the folding of GAPDH implies that endogenous metabolites may facilitate protein folding in vivo by interacting with partially unfolded intermediates.     

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

Background

The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics).

Methods

Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin–luciferase based real-time luminometry.

Results

Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%.Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux.Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers.

Conclusions

MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers.

General significance

Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.     

Monastrol inhibition of the mitotic kinesin Eg5

Monastrol is a small, cell-permeable molecule that arrests cells in mitosis by specifically inhibiting Eg5, a member of the Kinesin-5 family. We have used steady-state and presteady-state kinetics as well as equilibrium binding approaches to define the mechanistic basis of S-monastrol inhibition of monomeric human Eg5/KSP. In the absence of microtubules (Mts), the basal ATPase activity is inhibited through slowed product release. In the presence of microtubules, the ATPase activity is also reduced with weakened binding of Eg5 to microtubules during steady-state ATP turnover. Monastrol-treated Eg5 also shows a decreased relative affinity for microtubules under equilibrium conditions. The Mt.Eg5 presteady-state kinetics of ATP binding and the subsequent ATP-dependent isomerization are unaffected during the first ATP turnover. However, monastrol appears to stabilize a conformation that allows for reversals at the ATP hydrolysis step. Monastrol promotes a dramatic decrease in the observed rate of Eg5 association with microtubules, and ADP release is slowed without trapping the Mt.Eg5.ADP intermediate. We propose that S-monastrol binding to Eg5 induces a stable conformational change in the motor domain that favors ATP re-synthesis after ATP hydrolysis. The aberrant interactions with the microtubule and the reversals at the ATP hydrolysis step alter the ability of Eg5 to generate force, thereby yielding a nonproductive Mt.Eg5 complex that cannot establish or maintain the bipolar spindle.     

Identification and isolation of ATP transport protein in mycobacillin sensitive Aspergillus niger

The temperature sensitive release and uptake of ATP through theAspergillus niger G₃Br membrane vesicles followed saturation kinetics. Both the processes which occurred in the absence of mycobacillin were greatly enhanced by its presence. Liposomes prepared with antifilipin sterol and lipid showed the release and uptake of ATP in the presence of filipin, but no such uptake and release was seen with antimycobacillin sterol and lipid in the presence of mycobacillin. However the liposomes supplemented withAspergillus niger membranes protein (s) showed the release and uptake of ATP, implicating membrane protein as a carrier in the transport process.     

Reaction of a carbodiimide adduct of ATP at the active site of sarcoplasmic reticulum calcium ATPase

An adduct of a carbodiimide and ATP was synthesized from 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and the nucleotide. Despite its limited stability (t1/2 for hydrolysis of about 5 min at 25 degrees C), it was shown to react with and inactivate the calcium ATPase of sarcoplasmic reticulum in its vesicular, nonionic detergent-solubilized and purified forms. Saturation kinetics, with an ATP-EDC concentration dependence midpoint in the 10 microM range, were observed, suggesting an active-site affinity which is similar to ATP. The reaction was specific in that inactivation required reaction of about one adduct per ATPase. The modified enzyme could no longer be phosphorylated by ATP or Pi or hydrolyze p-nitrophenyl phosphate, but retained the ability to undergo the high-affinity calcium-dependent fluorescence change. It also bound trinitrophenyl-ADP and other nucleotides at least 10-fold more weakly than the unmodified ATPase. The inactivation reaction required the presence of Mg2+ and Ca2+ and was prevented by nucleotides such as ATP and ADP. For magnesium, the inactivation-enabling effect occurred with a midpoint of 3 mM. In the case of calcium, the transition resembled high-affinity binding in that it occurred cooperatively with a midpoint in the micromolar range. Higher [Mg2+] shifted this transition to higher [Ca2+]. Polyacrylamide gel electrophoresis (PAGE) demonstrated that the reaction converted the ATPase (Mr = 1.1 x 10(5)) to a species with an apparent Mr = (1.7-1.8) x 10(5). Since nonionic detergent-solubilized ATPase and purified ATPase gave similar results, intramolecular cross-linking is implicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of several nucleotides on the molecular state and catalytic activity of Thiobacillus novellus cytochrome c oxidase. ATP affects the oxidase uniquely.

The catalytic activity and molecular aspects of Thiobacillus novellus cytpchrome c oxidase were affected by ATP. The steady-state kinetics in the oxidation of ferrocytochrome c by the oxidase varied with the presence or absence of ATP; the [S]-v curve of the reaction was sigmoid in the absence of ATP whereas it was a Michaelis-Menten-type hyperbola in the presence of 700 microM ATP. The oxidase was a dimer of the minimal structural subunit consisting of one molecule each of two subunits in the presence of Tween 20 and in the absence of ATP. The dimer dissociated into monomers in the presence of 700 microM ATP. The trough at 452 nm seen in the second derivative absorption spectrum of the CO compound of the oxidase in the absence of ATP, a characteristic of the cytochrome a component of cytochrome aa3, dissappeared in the presence of 700 microM ATP. However, ADP, AMP, GTP, CTP and UTP had little affect on both the [S]-v curve and the molecular mass of the oxidase when used in place of ATP.     

Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells

Extracellular ATP is a potent autocrine/paracrine signal that regulates a broad range of liver functions through activation of purinergic receptors. In biliary epithelium, increases in cell volume stimulate ATP release through a phosphoinositide 3-kinase (PI3-kinase)-dependent mechanism. Because PI3-kinase also regulates vesicular exocytosis, the purpose of these studies was to determine whether volume-stimulated vesicular exocytosis contributes to cellular ATP release. In a human cholangiocarcinoma cell line, exocytosis was measured by using the plasma membrane marker FM1-43, whereas ATP release was assessed by using a luciferase-luciferin assay. Under basal conditions, cholangiocytes exhibited constitutive exocytosis at a rate of 1.6%/min, and low levels of extracellular ATP were detected at 48.2 arbitrary light units. Increases in cholangiocyte cell volume induced by hypotonic exposure resulted in a 10-fold increase in the rate of exocytosis and a robust 35-fold increase in ATP release. Both vesicular exocytosis and ATP release were proportional to cell volume, and both exhibited similar regulatory properties including: 1) dependence on intact PI3-kinase, 2) attenuation by inhibition of PKC, and 3) potentiation by activation of PKC before hypotonic exposure. These findings demonstrate that increases in cholangiocyte cell volume stimulate ATP release and vesicular exocytosis through similar regulatory paradigms. Functional interactions among cell volume, PKC, and PI3-kinase modulate exocytosis, thereby regulating ATP release and purinergic signaling in cholangiocytes. It is hypothesized that PKC is involved in the recruitment of a volume-sensitive vesicular pool to a readily releasable state.     

The Calculation of 3D Solubility Parameters Using Molecular Models

Abstract

In this paper we describe the use of molecular mechanics models to examine detailed intermolecular interactions within the liquid state of a common nonionic surfactant system, nonyl phenol ethoxylate (NPE). Using constant energy molecular dynamics simulations we have studied the relative strengths of dispersive interactions versus polar interactions and have estimated three dimensional solubility parameters for NPE systems as a function of temperature and ethylene oxide content. The predictions at 300 K are in good agreement with three dimensional solubility parameters predicted using group contribution tables. Models of the amorphous liquid state were represented by single molecular structures of NPE in a periodic cell. The solubility parameters predicted with these models were in good agreement with those values derived from models having eight NPE molecules packed into a cell with the exception of the electrostatic interactions, which are the most sensitive to system size effects.     

Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [(3)H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.     

Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release

Extracellular nucleotides such as ATP and UTP are released in response to mechanical stimulation in different cell systems. It is becoming increasingly evident that ATP release plays a role in autocrine and paracrine stimulation of osteoblasts. Mechanical stimulation, as shear stress, membrane stretch or hypo-osmotic swelling, as well as oscillatory fluid flow, stimulates ATP release from different osteoblastic cell lines. Human osteoblast-like initial transfectant (HOBIT) cells release ATP in response to mechanical stimulation. In the present study, we show that HOBIT cells are activated by nanomolar levels of extracellular ATP, concentrations that can be detected under resting conditions and increase following hypotonic shock. Cell activation by hypotonic medium induced intracellular Ca2+ oscillations, and Egr-1 synthesis and DNA-binding activity. Quinacrine staining of living, resting cells revealed a granular fluorescence, typical of ATP-storing vesicles. Monensin prevented quinacrine staining and considerably inhibited hypotonic-induced ATP release. Finally, elevated levels of cytosolic Ca2+ activated massive ATP release and a dose-dependent loss of quinacrine granules. The contribution of a vesicular mechanism for ATP release is proposed to sustain paracrine osteoblast activation.     

Heparan sulfate modulates kinin release by Trypanosoma cruzi through the activity of cruzipain.

Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K(i app)) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans.     

P-Glycoprotein kinetics measured in plasma membrane vesicles and living cells

P-glycoprotein (MDR1, ABCB1) is an ATP-dependent efflux transporter of a large variety of compounds. To understand P-glycoprotein in more detail, it is important to elucidate its activity in the cellular ensemble as well as in plasma membrane vesicles (under conditions where other ATP dependent proteins are blocked). We measured P-glycoprotein activity in inside-out vesicles formed from plasma membranes of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) for comparison with previous measurements of P-glycoprotein activity in living NIH-MDR1-G185 cells. In plasma membrane vesicles activity was measured by monitoring phosphate release upon ATP hydrolysis and in living cells by monitoring the extracellular acidification rate upon ATP synthesis via glycolysis. P-glycoprotein was stimulated as a function of the concentration with 19 structurally different drugs, including local anesthetics, cyclic peptides, and cytotoxic drugs. The concentrations of half-maximum P-glycoprotein activation, K1, were identical in inside-out plasma membrane vesicles and in living cells and covered a broad range of concentrations (K1 approximately (10(-8)-10(-3)) M). The influence of the pH, drug association, and vesicle aggregation on the concentration of half-maximum P-glycoprotein activation was investigated. The turnover numbers in plasma membrane vesicles and in living cells were also approximately identical if the latter were measured in the presence of pyruvate. However, in the absence of pyruvate they were higher in living cells. The rate of ATP hydrolysis/ATP synthesis decreased exponentially with decreasing free energy of drug binding from water to the transporter, DeltaG0(tw)(1) (or increasing binding affinity). This suggests that drug release from the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.     

Directional memory and caged dynamics in cytoskeletal remodelling

We report directional memory of spontaneous nanoscale displacements of an individual bead firmly anchored to the cytoskeleton of a living cell. A novel method of analysis shows that for shorter time intervals cytoskeletal displacements are antipersistent and thus provides direct evidence in a living cell of molecular trapping and caged dynamics. At longer time intervals displacements are persistent. The transition from antipersistence to persistence is indicative of a time-scale for cage rearrangements and is found to depend upon energy release due to ATP hydrolysis and proximity to a glass transition. Anomalous diffusion is known to imply memory, but we show here that memory is attributed to direction rather than step size. As such, these data are the first to provide a molecular-scale physical picture describing the cytoskeletal remodelling process and its rate of progression.     

The effects of octylglucoside on the interactions of chloroplast coupling factor 1 (CF1) with adenine nucleotides

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg-specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982) Biochemistry, 21, 6144-6152], on the interactions of the enzyme with adenine nucleotides have been studied. 1. OcGlc specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5'[beta, gamma-imido]triphosphate (AdoPP[NH]P) from the tight-sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc. 2.OcGlc-induced inactivation of CF1-ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP X CF1 is rapidly inactivated while ATP X CF1 and AdoPP[NH]P X CF1 are slowly inactivated by OcGlc in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1 is inhibited by OcGlc, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight-sites protects the enzyme against OcGlc-induced inactivation. 3. Mg ions specifically inhibit the release of bound ADP and the OcGlc-induced inactivation of CF1. High concentrations of medium ATP and ADP (K50 = 100 microM) also inhibit the OcGlc-induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides. 4. Mg-ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight-sites under these conditions where medium ATP is rapidly hydrolyzed. Mg-ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate. 5. It is suggested that: (a) ATP and ADP bind to the same tight-sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1 contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight-sites by cooperative interactions. (c) Mg-ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight-site. The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1 are discussed.