Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.     

Extraction and determination of adenosine 5'-triphosphate in bovine milk by the firefly luciferase assay

The release of ATP from somatic cells in milk with the detergent Triton X-100 was optimized for assay with firefly luciferase. A small volume of milk (40 microliters) is added to 0.8 ml of 0.2% Triton X-100 in 100 mM Tris, 4 mm EDTA, pH 7.8. After approximately 1 min, 0.2 ml of luciferase reagent is added and the emission of light is measured in a luminometer. Results are calibrated with an ATP standard. This single method gave high yields of ATP from somatic cells in milk without interference from bacterial ATP. Extracts could be stored or transported prior to assay without deterioration of results. A close correlation was found between somatic cell count and ATP in milk samples collected at a farm as well as in milk samples from a cow with experimental mastitis. Results are promising for future use for diagnosis of mastitis but further work and field testing has to be done before it can be used on a wider scale.     

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.

     

Interaction between collagen and adenosine 5'-triphosphate.

A quantitative study was made of the adsorption of adenosine 5′-triphosphate (ATP) on collagen by following the change in the absorbance at 258 nm of ATP in the soaking solution. The amount of ATP adsorbed decreased exponentially with the increase of pH up to pH 8 and fell off more rapidly at higher pH values. At a given pH, when the concentration of ATP was increased, the amount of ATP adsorbed increased following the pattern of a Langmuir isotherm. The adsorption was independent of the cation present. The adsorption of adenosine 5′-diphosphate was essentially the same as that for ATP. For tendons deposited with calcium phosphate, the amount of ATP adsorbed decreased compared to natural tendons. The adsorption of ATP on collagen fibers inhibited the contraction caused by calcium chloride, calcium bromide, and lithium bromide. In solution, ATP had very little effect on the denaturation of acid-soluble collagen caused by calcium chloride.     

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.     

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.     

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.     

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.     

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.     

Alpha-crystallin regions affected by adenosine 5'-triphosphate identified by hydrogen-deuterium exchange

ATP interaction with lens alpha-crystallins leading to enhanced chaperone activity is not yet well understood. One model for chaperone activity of small heat shock proteins proposes that ATP causes small heat shock proteins to release substrates, which are then renatured by other larger heat shock proteins. A similar role has been proposed for ATP in alpha-crystallin chaperone activity. To evaluate this model, ATP-induced structural changes of native human alpha-crystallin assemblies were determined by hydrogen-deuterium exchange. In these experiments, hydrogen-deuterium exchange, measured by mass spectrometry, gave direct evidence that ATP decreases the accessibility of amide hydrogens in multiple regions of both alphaA and alphaB. The surface encompassed by these regions is much larger than would be shielded by a single ATP, implying that multiple ATP molecules bind to each subunit and/or ATP causes a more compact alpha-crystallin structure. Such a conformational change could release a bound substrate. The regions most affected by ATP are near putative substrate binding regions of alphaA and alphaB and in the C-terminal extension of alphaB. The widespread decrease in hydrogen-deuterium exchange with particularly large decreases near substrate binding regions suggests that ATP releases substrates via both direct displacement and a global conformational change.     

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.     

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.     

Effect of adenosine 5'-triphosphate and adenosine 5'-diphosphate on the oxidation of cytochrome c by cytochrome c oxidase

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and inorganic pyrophosphate partially inhibit the oxidation of exogenous cytochrome c by cytochrome c oxidase of submitochondrial particles (with or without detergent treatment) or by a purified preparation when it is assayed polarographically in buffers of nonbinding ions at pH 7.8. ATP is somewhat more inhibitory than ADP. The inhibition is never greater than 50%, and it is always less than an equal concentration of Mg2+ ions is present or when the assays are run at pH 6. In contrast, the effect of ATP, ADP, and pyrophosphate on oxidase assays run spectrophotometrically is a similar slight stimulation of the oxidase of submitochondrial particles treated with deoxycholate and little or no effect on purified oxidase. The reaction of the oxidase of submitochondrial particles with the endogenous cytochrome c is stimulated by the nucleotides, as is the reduced nicotinamide adenine dinucleotide (NADH) oxidase activity. The observations can be explained by binding of ATP, ADP, or pyrophosphate to cytochrome c so that the formation of an especially reactive combination of cytochrome c and cytochrome oxidase previously postulated [Smith, L., Davies, H. C., & Nava, M. E. (1979) Biochemistry 18, 3140] is prevented. The data give no evidence that respiration via cytochrome c oxidase is regulated physiologically by direct effects of ATP or ADP on its activity.     

Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate

The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.