Interactions between the mitochondrial adenosinetriphosphatase and periodate-oxidized adenosine 5'-triphosphate, an affinity label for adenosine 5'-triphosphate binding sites

Periodate-oxidized ATP (o-ATP) was prepared as an affinity label of nucleotide binding sites on the chloroform-released ox heart mitochondrial ATPase. In the presence of MgSO4, o-ATP is a substrate for the ATPase. It can act as a reversible, competitive inhibitor of ATPase activity and can also induce an irreversible inhibition of ATPase activity. In parallel with the irreversible inhibition, covalent incorporation of [3H]o-ATP occurs. ATPase has about 1.05 mol of o-ATP bound per mol of ATPase when the enzyme is 50% inhibited. Most of the covalently bound o-ATP is associated with the alpha and beta subunits and is equally distributed between them. The incorporation of o-ATP into the ATPase is reduced, and the irreversible inhibition induced by o-ATP can be prevented totally by MgADP, MgATP, EDTA/ATP, or EDTA. The location, number, and the functional significance of the o-ATP binding sites are discussed. o-ATP can decompose to form an adenosine-containing compound and the tripolyphosphate anion in a beta-elimination reaction mechanism. The structures of the adenine-containing compound and its borohydride reduction product were determined. The adenine-containing elimination product inhibited the mitochondrial ATPase activity at a rate greater than that observed with o-ATP. The nature and mechanism of the inhibition of ATPase activity exerted by o-ATP and the elimination product were examined. The significance of the beta-elimination reaction to the use of periodate-oxidized nucleotides as affinity labels of nucleotide binding sites on other proteins is discussed.     

Interaction of prostaglandins and adenosine 5'-triphosphate in the noncholinergic neurotransmission in rabbit detrusor.

Part of the excitatory transmission in rabbit detrusor is noncholinergic and nonadrenergic, and prostaglandins (PGs) and adenosine 5'-triphosphate (ATP) have been implicated in this transmission. The present experiments investigate the possibility of an interaction between PGs and ATP in rabbit detrusor. Indomethacin (2.8 muM) depressed the contraction produced by ATP although it did not antagonize the contraction produced by carbachol. Treatment of detrusor strips with 1.5 mM ATP depressed the frequency response curve in field stimulated tissues. This depression was additive with that produced by atropine. In the present experiments indomethacin did not significantly augment the effect of desensitization with ATP. It is suggested that the atropine-resistant neurotransmission in rabbit detrusor may involve both ATP and PGs acting in cooperation.     

5’-三磷酸腺苷结晶热力学

5’-三磷酸腺苷（5’-ATP）是重要的生物活性物质,其结晶过程困难,是研究的热点。分别采用静态法和激光法测定了5’-ATP在不同温度的乙醇-水体系中的溶解度和介稳区宽度,并用溶解度模型——肌方程回归了溶解度数据,得到的溶解度关联方程为λ=19．034 3exp（-12．799w）,h：614．053exp（11．3106w）。研究表明．5’-ATP的溶解度随着温度的升高而升高,随着乙醇与水体积比r的升高而下降;其介稳区宽度随着温度的升高略有增大,随着乙醇与水体积比r的升高明显减小。这为5＇-三磷酸腺苷工业结晶装置及生产线的设计、工程放大和工业结晶生产操作提供了理论依据。     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate. I. Equilibrium model

The thermodynamic treatment of the disproportionation reaction of adenosine 5′-diphosphate to adenosine 5′-triphosphate and adenosine 5′-monophosphate is discussed in terms of an equilibrium model which includes the effects of the multiplicity of ionic and metal bound species and the presence of long range electrostatic and short range repulsive interactions. Calculated quantities include equilibrium constants, enthalpies, heat capacities, entropies, and the stoichiometry of the overall reaction. The matter of how these calculations can be made self-consistent with respect to both calculated values of the ionic strength and the molality of the free magnesium ion is discussed. The thermodynamic data involving proton and magnesium-ion binding data for the nucleotides involved in this reaction have been evaluated.     

Metabolism of exogenous uridine 5'-triphosphate,adenosine 5'-triphosphate and pyrophosphate by alkylsulfatase-producing bacteria

• 1.1. The uptake and metabolism of β-P³²-labelled UTP, ATP and uniformly labelled PPi was studied using bacteria washed twice with and immediately resuspended in a medium containing 0.031 M PO³⁻₄.
• 2.2. Under these conditions, little P³²-labelled PO³⁻₄ was detected extracellularly when the cells were exposed for 2–5 min to 5 × 10⁻⁵ M concentrations of the above effectors.
• 3.3. Cells of both Pseudomonas aeruginosa and Pseudomonas C₁₂B incorporated the P³²-label from all effectors under the above conditions.

     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Calorimetric analysis of aspartate transcarbamylase from Escherichia coli: binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate.

The binding of CTP and ATP to aspartate transcarbamylase at pH 7.8 and 8.5 at 25 degrees has been investigated by equilibrium dialysis and flow microcalorimetry. The binding isotherms for CTP at both pH 7.8 and 8.5 and ATP AT PH 8.5 can be fit by a model which assumes three tight, three moderately tight, and six weak binding sites. The binding isotherms for ATP at pH 7.8 are best fit by a model which assumes six tight and six weaker sites. Both finite differenceH binding and finite differenceS binding are negative for both nucleotides at both pH values, so that the binding is enthalpy driven. For both nucleotides, finite differenceH is the same for the first two classes of binding sites, implying that the difference in the dissociation constants of these two classes of sites is the result of entropic effects. Direct pH measurements and calorimetric measurements in two buffers with very different heats of ionization (Tris and Hepes) indicate that the binding of both nucleotides is accompanied by the binding of protons. In the pH range 6.7-8.4, the number of moles of protons bound per mole of nucleotide increases as the pH decreases.     

Standard thermodynamic formation properties for the adenosine 5'-triphosphate series.

The criterion for chemical equilibrium at specified temperature, pressure, pH, concentration of free magnesium ion, and ionic strength is the transformed Gibbs energy, which can be calculated from the Gibbs energy. The apparent equilibrium constant (written in terms of the total concentrations of reactants like adenosine 5'-triphosphate, rather than in terms of species) yields the standard transformed Gibbs energy of reaction, and the effect of temperature on the apparent equilibrium constant at specified pressure, pH, concentration of free magnesium ion, and ionic strength yields the standard transformed enthalpy of reaction. From the apparent equilibrium constants and standard transformed enthalpies of reaction that have been measured in the adenosine 5'-triphosphate series and the dissociation constants of the weak acids and magnesium complexes involved, it is possible to calculate standard Gibbs energies of formation and standard enthalpies of formation of the species involved at zero ionic strength. This requires the convention that the standard Gibbs energy of formation and standard enthalpy of formation for adenosine in dilute aqueous solutions be set equal to zero. On the basis of this convention, standard transformed Gibbs energies of formation and standard transformed enthalpies of formation of adenosine 5'-trisphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine at 298.15 K, 1 bar, pH = 7, a concentration of free magnesium ions of 10(-3) M, and an ionic strength of 0.25 M have been calculated.     

Histidine starvation and adenosine 5'-triphosphate depletion in chemotaxis of Salmonella typhimurium.

Starvation for histidine prevented tumbling in Salmonella typhimurium hisF auxotrophs, including constantly tumbling strains with an additional mutation in cheB or cheZ. However, histidine-starved cheZs hisF strains were not defective in flagellar function or the tumbling mechanism since freshly starved auxotrophs tumbled in response to a variety of repellents. Tumbling in histidine-starved S. typhimurium could be restored in 13 s by addition of adenine or in 4 min by addition of histidine. Chloramphenicol did not prevent restoration of tumbling by these substances. Assays of adenosine 5'-triphosphate were performed based upon previous demonstration of adenine depletion in hisF auxotrophs starved for histidine. The adenosine 5'-triphosphate concentration dropped rapidly during the course of starvation, falling to less than 5% of the initial level as the cells ceased tumbling entirely. The change to smooth motility was prevented by 2-thiazolealanine, which inhibits phosphoribosyltransferase, thereby preventing adenine depletion during histidine starvation. These results suggest that an adenosine 5'-triphosphate deficiency was responsible for the change in tumbling frequency.     

Microscopic counting and adenosine 5'-triphosphate measurement in determining microbial growth in soils.

A microscopic technique utilizing dispersion of fungal hyphae in a Waring blender, filtration through membrane filters (Nucleopore Corp.), and counting on a fluorescence microscope was developed for counting fungal hyphal biomass. Nonfluorescent staining techniques of the soil-filter preparation did not give quantitative recoveries. Water-soluble aniline blue, which binds to the beta-1,3-glucans of the fungal cell wall, made visualization of the hyphae by fluorescence possible. A range of fungi added to soil were quantitatively recovered. Adenosine 5'-triphosphate (ATP) was extracted from soil by lysis of the organisms with CHCl(3) in NaHCO(3), which prevented adsorption of the organic phosphorus to the soil colloids. Centrifugation and removal of CHCl(3) was followed by dilution with pH 7.8 tris(hydroxymethyl)aminomethane buffer. ATP concentrations were measured by using the luciferase-luciferin light reaction. Since NaHCO(3) interfered to some extent with this reaction, the standards were made up in equivalent mixtures of tris(hydroxymethyl)aminomethane buffer and NaHCO(3). Recovery of ATP was rapid and quantitative in a range of soils. Measurement of the ATP and bacterial and fungal numbers in an incubated soil showed that fungal and bacterial population increases were delayed by phosphorus deficiency. Microbial populations were not affected at a later date. The ATP content of the soil system was reduced by phosphorus deficiency throughout the incubation period. This indicated that ATP could be altered without major changes in the microbial populations.     

Stimulus-secretion coupling in platelets. Effects of drugs on secretion on adenosine 5'-triphosphate.

The mechanism of stimulus-secretion coupling in platelets was investigated by observing the effects of drugs on the kinetics on ATP secretion induced by either thrombin or the divalent cation ionophore A23187. The actual secretion is the same with either of these agents, since the rate constants and activation energies of secretion are the same and since drugs that affect the final, enzyme-independent steps of thrombin-induced secretion have the same effect on ionophore-induced secretion. Drugs that affect early steps of thrombin-induced secretion have no effect on ionophore-induced secretion. Drugs that act through cAMP (PGE1, theophylline, dibutyryl-cAMP) slow an early step in the mechanism of thrombin-induced secretion and completely block at higher levels, with the required concentration of inhibitor dependent on thrombin concentration. The inhibition of rate appears to be all-or-none, with no intermediate rates observed. By replacing thrombin with trypsin, which makes it possible to observe a complete change in rate-determining step from an enzyme-dependent to an enzyme-independent platelet step, it was found that these drugs slow the rate only when the enzyme-independent step is rate determining. These drugs have no effect on A23187-induced secretion. It was concluded that cAMP inhibits at a step after the enzyme step but before the final step by interfering with transmission of the stimulus-secretion coupling signal. Disruption of microfilament function by cytochalasin B (10 muM) accelerates the rate of secretion induced by either thrombin or ionophore. The microtubule agents colchicine, vinblastine, and vincristine had effects only at concentrations above those usually considered necessary for the specific inhibition of microtubule function. Drugs that inhibit prostaglandin synthesis (aspirin, indomethacin, eicosatetraynoic acid), drugs that block ATP production (antimycin A, deoxyglucose), or several other drugs previously reported to inhibit platelet function had no effect on secretion.     

Complexes of cobalt (II) and manganese (II) with adenosine 5'-diphosphate and adenosine 5'-triphosphate. A circular dichroism study.

For studies of interactions between Co2+ and adenosine 5'-diphosphate or adenosine 5'-triphosphate (ADPH4+ and ATPH5+ in strongly acidic medium) visible circular dichroism (d-d transitions of Co2+) and ultraviolet circular dichroism (adenine transitions) have proven to be very sensitive to structural changes. Drastic variation of spectra as a function of pH and concentration enabled us to show the existence of various species, to state their stoichiometry and eventually, their self-association. With ATPH22-, C.D. results are in agreement with recent N.M.R. results. With ligands bearing three negative charges, complexes (1 metal:2 nucleotides)n are formed in which bases of the two nucleotides of the molecule are self-associated. With ADP3-, the visible C.D. spectrum of this complex is intense and hides the spectra of the complexes formed with other protonated species of ADP; this self-associated complex is detected up to a lower limit of 5 X 10(-4) M concentration. With ATPH3-, a complex exhibiting the same characteristics as the one with ADP3- is formed but in about twenty times less amount which explains why it was not detected by potentiometry. With 0.1 M ATP4-, dimeric (or polymeric) complexes, of 1:2 and 1:1 stoichiometry are observed. With 0.01 M ATP4-, 1:1 monomeric and 2:1 dimeric (or polymeric) complexes are detected. The interactions between Mn2+ ions and ADP or ATP have been studied by C.D. on the UV range. The same species as with Co2+ ions have been found but the 1:2 complex formation with ADP3- was shown to occur to a lesser extent and was not observed below a 10(-2) M ADP concentration.