Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Arsenylation of nucleoside diphosphates in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores catalyze the formation of ADP-arsenate during illumination with ADP, Mg²⁺ and arsenate. The reaction was measured with (1) firefly luciferase, (2) a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase, and (3) a glass electrode. ADP-arsenate hydrolyzed spontaneously with rate constants ranging from 14 to 43 min^?1. Magnesium, arsenate and phosphate accelerated hydrolysis of ADP-arsenate. From a comparison of the three methods, with ADP as the substrate, it is estimated that φ_R (i.e., the ratio between the quantum yields of ADP-arsenate and ATP for light emission from luciferase) is 0.19–0.23. Furthermore, arsenylation rates were 46–52% of phosphorylation rates in experiments with 30 μ M ADP and 0.8 mM arsenate or phosphate. Similarly, the V_app for arsenylation of GDP or IDP was 47–59% of the V_app for phosphorylation during measurements in the presence of 1 mM arsenate or phosphate. The K_app(GDP) was higher during arsenylation than during phosphorylation; the K_app(IDP) was about the same during arsenylation as during phosphorylation. It is suggested that a shift in the equilibrium of substrates and products on the enzyme, toward hydrolysis, is the main cause of the relatively low arsenylation rates.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Decay of membrane potential under phosphorylating conditions in chloroplasts with in vivo activated ATPase

(1) The relation between the membrane potential and phosphorylation was studied in chloroplasts rapidly prepared from illuminated spinach leaves (light chloroplasts) and from dark-adapted leaves (dark chloroplasts). Light chloroplasts had a higher ATP hydrolysis activity than dark chloroplasts. (2) In the presence of ADP or ATP, a rapidly decaying phase of the field-indicating 518 nm absorbance change with a half-time of 15 ms became apparent in addition to the slow phase with a half-time of more than 300 ms in either type of chloroplast. Under these conditions, light chloroplasts showed a larger rapid phase than dark chloroplasts. (3) The rapid phase was suppressed by dicyclohexylcarbodiimide and was assumed to reflect the dissipation of membrane potential due to proton movements inside the CF₁-CF₀ ATP synthetase. (4) A model for the proton movement in ATP synthetase is proposed.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF₁ were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl₂, 1 mol of ATP or FTP is bound to the latent enzyme, with K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K₁ = 2.4 · 10⁴ M^?1 · s^?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k_?1 = 3 · 10^?3 s^?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10⁶ M^?1 · s^?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-P_i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P_i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P_i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P_i-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.     

Acid phosphatases of rabbit spermatozoa: II. Partial purification and biochemical characterization of the multiple forms of rabbit spermatozoan acid phosphatase

Five acid phosphatases, S4, S3, S2, Szn and S1 (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) of ejaculated rabbit spermatozoa were either partially purified by DEAE-Sephadex column chromatography or prepared by specific extraction methods.The pH optimum of S4 was 6.0–6.5 in acetate buffer and 7.0 in Tris-HCl buffer; the pH optima of S3, S2, Szn, and S1 were 4.5, 5.5., 6.0 and 5.2, respectively, in acetate buffer. The apparent molecular weights of S3, S, Szn and S1, determined by disc gel electrophoresis, were 123 000, 86 000, 64 000 and 45 000–49 000, respectively. Incubation with neuraminidase did not alter the electrophoretic mobilities of any of the enzymes.Ten natural phosphoric esters were tested as substrates. S4 preferentially hydrolyzed ATP, ADP, PP_i and 3′-AMP. S3 hydrolyzed only β-glycerophosphate and glucose 6-phosphate to a significant extent. S2 hydrolyzed β-glycerophosphate, glucose 1-phosphate, the phosphoproteins, casein and phosvitin. S1 hydrolyzed ADP and β-glycerophosphate most readily. Szn may be an ATPase since it exhibits very high Zn²⁺-stimulated against ATP.These characteristics combined with the effects of NaF, ZnCl₂, l-(+)-tartaric acid, and formaldehyde on the activity of each partially purified enzyme with α-naphthyl phosphate as substrate indicate that these phosphatases are structurally and functionally different.     

Effects of mutations in the beta subunit hinge domain on ATP synthase F1 sector rotation: interaction between Ser 174 and Ile 163

A complex of γ, ε, and c subunits rotates in ATP synthase (F_oF₁) coupling with proton transport. Replacement of βSer174 by Phe in β-sheet4 of the β subunit (βS174F) caused slow γ subunit revolution of the F₁ sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including β-sheet4 and α-helixB predicted that the mutant βPhe174 residue undergoes strong and weak hydrophobic interactions with βIle163 and βIle166, respectively. Supporting this prediction, the replacement of βIle163 in α-helixB by Ala partially suppressed the βS174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of βIle166 by Ala lowered the revolution speed and ATPase activity to the same levels as in βS174F. Consistent with the weak hydrophobic interaction, βIle166 to Ala mutation did not suppress βS174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/α-helixB/loop/β-sheet4, βPhe148-βGly186] as to driving rotational catalysis is discussed.     

Dynamic affinity chromatography of heavy meromyosin subfragment-1

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative 3ave been chromatographed on immobilized ATP, ADP and adenosine 5′-(β,γ-imino)triphosphate affinity chromatography columns, in the presence and in the absence of Mg²⁺ or Ca²⁺. Splitting of bound ATP was followed by using [γ-^{3 2}P]ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5′(β,γ-imino)triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the P_i-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.     

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.     

The activity of phosphate-dependent glutaminase from the rat small intestine is modulated by ADP and is dependent on integrity of mitochondria

The effect of adenine nucleotides and phosphate on rat small intestine phosphate-dependent glutaminase (PDG) activity was investigated in intact mitochondria. Disruption of the integrity of mitochondria by sonication or freeze-thawing resulted in loss of enzyme activity. ADP was the strongest adenine nucleotide activator of the enzyme giving a V_max that was over 5-fold of that for AMP or ATP. The sigmoid activation curve of PDG by ADP became hyperbolic in presence ATP. ADP also lowered the K_m for glutamine and increased V_max and these effects were further enhanced by the presence of ATP. Activation of PDG by phosphate and ADP was not completely additive suggesting some antagonism between the activators. There was no clear relationship between changing ATP/ADP ratios and PDG activity in presence of a constant concentration of phosphate. However, ratios of approximately 1:4 and 4:1 gave the highest and lowest activities, respectively. The pH dependence of PDG activity was affected by phosphate concentration and results suggest that the divalent ion is the activating species.     

Loose and tight binding of adenine nucleotides by membrane-associated chloroplast ATPase

Steady-state binding of adenine nucleotides by thylakoid membranes is measured by employing a centrifugation technique. By this method tightly bound nonexchangeable nucleotides can be discriminated from loosely bound, exchangeable nucleotides. Nucleotide binding requires membrane energization and is highly specific for medium ADP. In illuminated chloroplasts almost no exogenous AMP and only some ATP are incorporated, most being recovered as tightly bound nucleotides. In light-triggered chloroplasts, however, which are capable of hydrolyzing ATP, a high level of exchangeable nucleotides is found on the membranes. The sum of tightly bound and loosely bound nucleotides originating from medium ADP is about one per CF₁. The ratio between them decreases with increasing proton-motive force. Exchangeable nucleotides most probably represent the ligands involved in the catalytic process, as suggested from substrate specificity and the effect of a competitive inhibitor of photophosphorylation, naphthoyl ADP. This compound in a low concentration range supresses loose binding but not tight binding of medium ADP. Under phosphorylating conditions (presence of ADP, P_i and light), some of the tightly bound nucleotides exist as ATP even in the presence of a hexokinase system. The results are discussed in the context of the regulation of chloroplast ATPase by tight nucleotide binding.     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.     

Magnesium spin state determines the energetics of an adenosinetriphosphate molecule

The molecular dynamics method (density functional theory) DFT:B3LYP (6-3IG** basis set, t = 310 K) was used to study interactions between a molecule of adenosinetriphosphate (ATP) (ATP subsystem) and the [Mg(H₂O)₆]²⁺ magnesium cofactor (Mg subsystem) in an aqueous medium simulated by 78 water molecules in the singlet (S) and triplet (T) states. Potential energy surfaces (PESs) for the S (lowest in energy) and T states (highest in energy) are significantly separated in space. Motion along them directs the Mg complex either to oxygen atoms of the γ-β-phosphate groups (O1–O2) (S state of PES) or to oxygen atoms of the β-α-phosphate groups (O2–O3) (T state of PES). Chelation of the γ-β- and β-α-phosphates leads to formation of a stable low-energy ([Mg(H₂O)₄-(OI-O2)ATP]^2?) complex or a metastable high-energy ([Mg(H₂O)₂-(O2–O3)ATP]^2?) complex, respectively, which differ in number of water molecules surrounding the Mg atom. Intersection of two T PESs is accompanied by formation of an unstable state characterized by redistribution of spins between the Mg and ATP subsystems. This state, being sensitive to interaction with the Mg nuclear spin (²⁵Mg), induces an unpaired electron spin, which initiates the ATP cleavage by the ion-radical mechanism, yielding a reactive ion radical of adenosinemonophosphate (·AMP^?), which was earlier found experimentally by the method of chemically induced dynamic nuclear polarization (CIDNP). Biological aspects of the results obtained are discussed.     

A simple and defined method to study calcification by isolated matrix vesicles effect of ATP and vesicle phosphatase

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 μg of protein, were incubated for 2 h in a ⁴⁵Ca-labelled solution with 2.2 mM Ca²⁺, 1.6 mM PO₄^3? and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO₄ hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca²⁺ or Mg²⁺ was found to stimulate matrix vesicle. ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, β-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, veiscles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.