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PubMed1989

Anthraniloyl ATP, a fluorescent analog of ATP, as a substrate for dynein ATPase and flagellar motility.

We synthesized an anthraniloyl ATP (ant-ATP), which has a fluorescent anthraniloyl moiety at the OH group of ribose, to elucidate the mechanism of flagellar bend formation and its propagation in relation to the mechanochemical cycle of dynein ATPase. This fluorescent analog of ATP was efficiently hydrolyzed by 21 S dynein from sea urchin sperm flagella with Km = 7.6 microM, whereas the Km was 12 microM when ATP was used as the substrate. Similar Vmax values were obtained with both ATP and ant-ATP. Inhibition of the hydrolysis of ant-ATP by vanadate was a little smaller than that with ATP. Photosensitized cleavage of 21 S dynein heavy chains in the presence of ant-ATP and vanadate was also a little less efficient than that in the presence of ATP and vanadate. Ant-ATP also induced the disintegration of the trypsin-treated axoneme and the motility of demembranated sperm in a manner similar to ATP. When ATP was used as a substrate for the demembranated sperm, the apparent Michaelis constant for beat frequency (Km f) was 0.22 mM and the maximum frequency (fmax) was 36 Hz, whereas Km f) was 0.14 mM and fmax was 20 Hz for ant-ATP. Thus ant-ATP could be an efficient fluorescent analog of ATP for studying dynein ATPase and the mechanisms of flagellar motility.

Adenosine Triphosphatases↗

PubMed2005

Regulation of the ATP-sensitive K channel Kir6.2 by ATP and PIP(2).

ATP-sensitive K (K(ATP)) channels are blocked by ATP and activated by PIP(2). Both negatively-charged ligands are presumed to bind to positively-charged residues on the N-and C-termini of the channel's cytoplasmic domain. Evidence summarized here suggests that the channel's interaction with ATP and PIP(2) is regulated by separate groups of residues, involving both direct charge-charge interactions and allosteric effects. ATP interaction is regulated by R50 in the N-terminus and by K185, R192 and R201 in the C-terminus. R192 and R201 mutations decrease channel sensitivity to ATP, ADP and AMP to a similar extent, implying that they regulate interaction with either the alpha phosphate group, common to all three adenine nucleotides, or the adenosine moiety. K185 mutations, and to a lesser extent R50 mutations, decrease ATP and ADP sensitivity without markedly affecting AMP sensitivity, implying that they regulate interaction with the beta phosphate of ATP and ADP. In addition, when open probability decreases due to rundown, ATP sensitivity increases in R50, K185 and R192, but not in R201 mutants. Combining these observations with recent structural data, we hypothesize the following scenario: 1) the ATP binding site is located at the outside of the channel's cytoplasmic domain away from the pore. 2) When the channel is open, R50 and K185 interact directly with the beta phosphate of ATP, whereas R192, which appears to be removed from the ATP binding site, modulates the initial interaction with ATP allosterically. 3) When the channel closes, R201 is in position to interact with the alpha phosphate of ATP to stabilize the closed state. 4) PIP(2) also interacts with the channel's cytoplasmic domain, but at distinct positively-charged residues located above the ATP binding site and near to the plasma membrane. These residues include R54 in the N-terminus and R176, R177 and R206 in the C-terminus. Thus, the binding domains of ATP and PIP(2) in the N- and C-termini do not appear to overlap.

Adenosine Triphosphate↗

PubMed2003

Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed, pre-transition-state conformations.

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.

Adenosine Triphosphate↗

PubMed1998

Kinetics of the reactions of the Escherichia coli molecular chaperone DnaK with ATP: evidence that a three-step reaction precedes ATP hydrolysis.

The mechanism of the ATPase cycle of the 70-kDa Escherichia coli molecular chaperone DnaK was investigated by following ATP-induced changes in the tryptophan fluorescence of DnaK. Three steps in the cycle were investigated. (i) Stopped-flow experiments revealed that ATP induces a biphasic reduction in the tryptophan fluorescence of DnaK. The rate of the fast fluorescence transition exhibited a hyperbolic dependence on the ATP concentration, with a maximum rate equal to 56 (+/- 10) s-1 at 35 degrees C, whereas the rate of the slow fluorescence transition was nearly independent of the ATP concentration (4.2 +/- 0.2 s-1). These results are consistent with the three-step sequential reaction E + ATP<-->E-ATP<-->E*-ATP<-->E**-ATP prior to DnaK-catalyzed ATP hydrolysis, where the formation of a collisional complex (E-ATP) causes no change in fluorescence but is followed by two first-order transitions that reduce the fluorescence. (ii) The kinetics of ADP replacement from preformed DnaK-ADP complexes by ATP followed simple exponential kinetics, kADP = 0.038 (+/- 0.002) s-1 at 35 degrees C. The ADP off rate was reduced approximately 10-fold by inorganic phosphate (20 mM). (iii) Single-turnover experiments ([DnaK] = [ATP] = 1 microM) revealed a slow, first-order increase in tryptophan fluorescence [k(obs) = 0.0015 (+/- 0.0001) s-1, 37 degrees C] that was identical to the rate of DnaK-catalyzed ATP hydrolysis [k(hy) = 0.0014 (+/- 0.0001) s-1, 37 degrees C]. This slow increase in fluorescence is consistent with a E**-->E conformational transition. A model for the ATPase cycle of DnaK is proposed in which ATP has two distinct functions: ATP binding to the ATPase domain triggers two conformational transitions in a chaperone molecule, and ATP hydrolysis--the slow step in the reaction cycle--reverses the transitions.

Adenosine Diphosphate↗

PubMed2003

NADH supplementation decreases pinacidil-primed I K ATP in ventricular cardiomyocytes by increasing intracellular ATP.

1 The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current I(K(ATP)) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2 Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36-37 degrees C. Adenine nucleotides were determined by HPLC. 3 Under physiological conditions (4.3 mM ATP in the pipette solution, ATP(i)) I(K(ATP)) did not contribute to basal electrical activity. 4 The ATP-dependent potassium (K((ATP))) channel opener pinacidil activated I(K(ATP)) dependent on [ATP](i) showing a significantly more pronounced activation at lower (1 mM) [ATP](i). 5 Supplementation of cardiomyocytes with 300 micro g ml(-1) NADH (4-6 h) resulted in a significantly reduced I(K(ATP)) activation by pinacidil compared to control cells. The current density was 13.8+/-3.78 (n=6) versus 28.9+/-3.38 pA pF(-1) (n=19; P<0.05). 6 Equimolar amounts of the related compounds nicotinamide and NAD(+) did not achieve a similar effect like NADH. 7 Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6+/-1.88 nmol mg(-1) protein versus control: 35.4+/-2.57 nmol mg(-1) protein, P<0.000005). 8 These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of I(K(ATP)) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9 Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.

Action Potentials↗

PubMed2002

Open state destabilization by ATP occupancy is mechanism speeding burst exit underlying KATP channel inhibition by ATP.

The ATP-sensitive potassium (K(ATP)) channel is named after its characteristic inhibition by intracellular ATP. The inhibition is a centerpiece of how the K(ATP) channel sets electrical signaling to the energy state of the cell. In the beta cell of the endocrine pancreas, for example, ATP inhibition results from high blood glucose levels and turns on electrical activity leading to insulin release. The underlying gating mechanism (ATP inhibition gating) includes ATP stabilization of closed states, but the action of ATP on the open state of the channel is disputed. The original models of ATP inhibition gating proposed that ATP directly binds the open state, whereas recent models indicate a prerequisite transition from the open to a closed state before ATP binds and inhibits activity. We tested these two classes of models by using kinetic analysis of single-channel currents from the cloned mouse pancreatic K(ATP) channel expressed in Xenopus oocytes. In particular, we combined gating models based on fundamental rate law and burst gating kinetic considerations. The results demonstrate open-state ATP dependence as the major mechanism by which ATP speeds exit from the active burst state underlying inhibition of the K(ATP) channel by ATP.

Adenosine Triphosphate↗

PubMed1977

Kinetic studies on the ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase. Evidence for the K.S.T. mechanism with two enzyme-ATP complexes and two phosphorylated intermediates of high-energy type.

The kinetic properties of the [3H]ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3.6.1,3] were investigated, using NaI-treated microsomes from bovine brain, and the following results were obtained. 1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45 micronM free Mg2+, 100 mMNaCl, 80 micronM ATP, and 160 micronM ADP at pH 6.5 and 4-5 degrees. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction parameters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 micronM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM. The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants Km, for both were 43 micronM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 micronM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 micronM ATP. 2. The N+-dependent [3H]ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+-concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H]ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration. From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP + ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP (formula: see text) produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step (formula: see text), determined by a method previously reported by us.

Adenosine Diphosphate↗

PubMed2001

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes.

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.

Adenosine Triphosphate↗

PubMed2003

Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction.

The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 microM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 microM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.

Adenosine Triphosphate↗

PubMed2003

MCC-134, a single pharmacophore, opens surface ATP-sensitive potassium channels, blocks mitochondrial ATP-sensitive potassium channels, and suppresses preconditioning.

BACKGROUND: MCC-134 (1-[4-(H-imidazol-1-yl)benzoyl]-N-methylcyclobutane-carbothioamide), a newly developed analog of aprikalim, opens surface smooth muscle-type ATP-sensitive potassium (K(ATP)) channels but inhibits pancreatic K(ATP) channels. However, the effects of MCC-134 on cardiac surface K(ATP) channels and mitochondrial K(ATP) (mitoK(ATP)) channels are unknown. A mixed agonist/blocker with differential effects on the two channel types would help to clarify the role of K(ATP) channels in cardioprotection. METHODS AND RESULTS: To index mitoK(ATP) channels, we measured mitochondrial flavoprotein fluorescence in rabbit ventricular myocytes. MCC-134 alone had little effect on basal flavoprotein fluorescence. However, MCC-134 inhibited diazoxide-induced flavoprotein oxidation in a dose-dependent manner (EC(50)=27 micro mol/L). When ATP was included in the pipette solution, MCC-134 slowly activated surface K(ATP) currents with some delay (>10 minutes). These results indicate that MCC-134 is a mitoK(ATP) channel inhibitor and a surface K(ATP) channel opener in native cardiac cells. In cell-pelleting ischemia assays, coapplication of MCC-134 with diazoxide abolished the cardioprotective effect of diazoxide, whereas MCC-134 alone did not alter cell death. These results were reproducible in both rabbit and mouse myocytes. MCC-134 also attenuated the effect of ischemic preconditioning against myocardial infarction in mice, consistent with the results of cell-pelleting ischemia assays. CONCLUSIONS: A single drug, MCC-134, opens surface K(ATP) channels but blocks mitoK(ATP) channels; the fact that this drug inhibits preconditioning reaffirms the primacy of mitoK(ATP) rather than surface K(ATP), channels in the mechanism of cardioprotection.

Adenosine Triphosphate↗

PubMed1994

Role of extracellular ATP in cell-mediated cytotoxicity: a study with ATP-sensitive and ATP-resistant macrophages.

It has been proposed that extracellular ATP (ATPo) may function as a cytotoxic molecule in Ca(2+)-independent cell-mediated lysis by activating plasma membrane P2 purinergic receptors. In the present study the involvement of the P2z purinergic receptor in ATPo as well as cell-mediated lysis was investigated by using the J774 mouse macrophage cell line, which expresses this receptor, and a panel of J774-derived mutant cell clones selected for the lack of P2z receptor activity. We confirmed that the P2z receptor in J774 is associated with ATPo-induced colloido-osmotic lysis but not with apoptosis. Furthermore, we observed that the lack or the inhibition of the P2z purinergic receptor does not affect lytic activity mediated by different types of cytotoxic cell populations. These results on the whole indicate that the P2z receptor is involved in cell membrane damage induced by ATPo but not in cell-mediated cytotoxicity.

Adenosine Triphosphate↗

PubMed1983

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase.

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.

ATP Synthetase Complexes↗

PubMed1985

Relationships between the rate of synthesis of ATP and the concentrations of reactants and products of ATP hydrolysis in maize root tips, determined by 31P nuclear magnetic resonance.

Using 31P NMR spectroscopy, we have measured the rate of ATP synthesis, and the free concentrations of ATP, ADP, cytoplasmic Pi, and H+ in maize root tips under a wide range of conditions. We show that the ratio [ATP]/[ADP] in normoxic root tips is greater than 25. We found no simple relationship between the concentration of ATP and the rate of ATP synthesis: when the rate of ATP synthesis decreases in response to different treatments, the concentration of ATP can increase, decrease, or remain unchanged. Clear relationships were obtained, however, when the rate of synthesis of ATP was plotted against the logarithm of the ratio psi, defined as [ATP]/[ADP][Pi][H+]. Two curves were obtained, depending on which of two situations pertained. First, if mitochondrial ATP synthesis was inhibited, e.g., by KCN or hypoxia, ln psi decreased monotonically as rates of ATP synthesis decreased. The decrease in ln psi may account for decreases in the rates of biosynthetic reactions dependent on ATP, such as protein synthesis, as they approach equilibrium. Second, if consumption of ATP for biosynthetic reactions was inhibited, by treatment with succinate, ln psi increased as rates of ATP synthesis decreased. The increase in ln psi may account for decreases in the rate of ATP synthesis, as oxidative phosphorylation approaches equilibrium.

Adenosine Diphosphate↗

PubMed1993

Mg2+ and ATP or adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase.

ATP and Mg2+ caused a transient increase in the intrisinc fluorescence of Rubisco activase which was inhibited by the presence of ADP. Only minor changes in fluorescence were observed with ATP or Mg2+ alone. The fluorescence increase was stabilized by addition of an ATP regenerating system or by substitution of ATP with a non-hydrolyzable analog, adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S). The initial rate of increase in fluorescence also depended on the concentration of protein in a manner consistent with second-order kinetics. The concentration dependence for the effect of ATP gamma S was sigmoidal, although at pH 8 the half-saturation requirements for both ATP gamma S (12 microM) and Mg2+ (1.5 mM) were not too dissimilar to the binding affinities (6 microM and 2 mM, respectively) determined indirectly with the fluorescent probe, 1-anilinonapthalene-8-sulfonate. However, the concentration dependence of ATP was about 5-fold higher than its binding affinity, also sigmoidal and quite similar to the concentration responses of ATP hydrolysis and activation of Rubisco by the protein. These characteristics of the intrinsic fluorescence indicate that it monitors a conformational change in the protein occurring after binding of the nucleotide and associated with increased aggregation. Direct evidence of increased aggregation in the presence of Mg2+ and ATP or ATP gamma S was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogeneous and also varied with temperature. The increased aggregation of the protein resulted in altered kinetic properties. The ATP hydrolysis activity of the protein increased and the half-maximal ATP concentration decreased as the protein concentration was increased in the assay. Also, a brief pretreatment of the protein with ATP and Mg2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.

Adenosine Triphosphate↗

PubMed1998

Probing the ATP binding site of tubulin with thiotriphosphate analogues of ATP.

Tubulin assembly studies with GTP alpha S diastereoisomers have shown that there is stereoselectivity at the alpha-phosphate binding region of tubulin. GTP alpha S(Sp) bound tighter than GTP alpha S(Rp) and promoted nucleation and assembly better than GTP and GTP alpha S(Rp). ATP and dATP have been reported to bind weakly to tubulin and to be less effective than GTP and dGTP in promoting tubulin assembly. This study was done to learn if ATP alpha S(Sp) and dATP alpha S(Sp) are good promoters of tubulin assembly and to compare these ATP thiotriphosphate analogues to the corresponding GTP analogues in tubulin assembly. Studies were also done with ATP alpha S(Rp), GTP, ATP beta S(Sp) and ATP gamma S. At least three cycles of tubulin (25 microM) assembly-disassembly were found with 1 mM ATP alpha S(Sp) and dATP alpha S(Sp) and both nucleotides were incorporated and hydrolyzed in the polymers. Less dATP alpha S(Sp) (25 microM) than ATP alpha S(Sp) (100 microM) promoted assembly to 50% of the maximum value. The critical concentrations (Cc) for assembly with 1 mM nucleotide were low for ATP alpha S(Sp) (3 microM) and dATP alpha S(Sp) (2 microM) and compared favorably with GTP (5 microM), GTP alpha S(Sp) (2 microM) and dGTP alpha S(Sp) (1 microM). Both 1 mM ATP and dATP were poor promoters of tubulin assembly and were not detected in the polymers. The predominant structures induced by 1 mM (ATP alpha S(Sp) and dATP alpha S(Sp) were bundles of sheets and microtubules, which were more stable to the cold and to Ca(II) than microtubules assembled with GTP, ATP or dATP. ATP alpha S(Rp) (1 mM) did not promote assembly suggesting that there is stereoselectivity at the ATP alpha S alpha-phosphate binding region of tubulin as there is with GTP alpha S diastereoisomers. ATP alpha S(Sp) and dATP alpha S(Sp) mimic GTP alpha S(Sp) and dGTP alpha S(Sp) in tubulin assembly since all four nucleotides promote bundles of tubulin in buffer with glycerol, and the deoxy nucleotides have lower Cc, shorter lags and faster rates for tubulin assembly.

Adenosine Triphosphate↗

PubMed1994

Identification of an amino acid in the ATP binding site of Na+/K(+)-ATPase after photochemical labeling with 8-azido-ATP.

[alpha-32P]-8-N3-ATP, [2-3H]-8-N3-ATP, and non-radioactive 8-N3-ATP have been used as photoaffinity probes of the ATP binding site of dog kidney Na+/K(+)-ATPase. 8-N3-ATP has previously been shown to bind to Na+/K(+)-ATPase with high affinity, to be a substrate for Na+/K(+)-ATPase, and to inactivate the enzyme upon ultraviolet irradiation [Scheiner-Bobis, G., & Schoner, W. (1985) Eur. J. Biochem. 152, 739-746]. 8-N3-ATP competitively inhibits the high-affinity binding of [2,8-3H]-ATP to Na+/K(+)-ATPase with a Ki of 3.4 microM, which is comparable to the reported KD of 3.1 microM for the binding of 8-N3-ATP to the enzyme. The extent of inhibition of ATP hydrolysis by 8-N3-ATP was linearly correlated with the stoichiometry of covalent incorporation of 8-N3-ATP into Na+/K(+)-ATPase up to about 50% inhibition of activity; however, the linkage between the protein and 8-N3-ATP was unstable, and the maximum incorporation of 8-N3-ATP was less than the nucleotide binding capacity of the protein. After photolysis with ultraviolet light, 8-N3-ATP was specifically incorporated into the carboxy-terminal 58-kDa fragment of the alpha-subunit of Na+/K(+)-ATPase generated by limited trypsin digestion in the presence of KCl, and the beta-subunit was not labeled. 8-N3-ATP-labeled Na+/K(+)-ATPase was digested with trypsin, and a single peak containing the nucleotide was identified after HPLC fractionation of the digest.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1985

Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange.

The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO3 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. Carbonyl cyanide m-chlorophenylhydrazone and NH4Cl (uncouplers of photophosphorylation) and phloridzin (an energy-transfer inhibitor) also lower the rate of ATP-H2O back-exchange; they too are found to act by decreasing the turnover of the ATP pool, not the extent of exchange per reacting ATP molecule. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form. The mode of NH4Cl inhibition of the ATP synthesis reaction, therefore, is not through a change in the partitioning of the E X ATP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1992

Extracellular cations modulate the ATP sensitivity of ATP-K+ channels in rat ventromedial hypothalamic neurons.

ATP-sensitive K+ (ATP-K+) channels underlie the glucose-sensing nature of pancreatic beta-cells by way of their inhibition by intracellular ATP. Recently it has been proposed that ATP-K+ channels have a similar function in certain hypothalamic neurons that become excitable in raised concentrations of extracellular glucose. The aim of this study was to assess the ATP sensitivity of ATP-K+ channels in inside-out membrane patches excised from glucose-sensing neurons that were acutely isolated from the ventromedial nucleus of rat hypothalamus. ATP-K+ channels were less sensitive to ATP in neurons than in other tissues. Moreover, the sensitivity of neuronal ATP-K+ channels to inhibition by intracellular ATP was modulated by extracellular cations. Under physiological ionic gradients (i.e. high extracellular Na+ and low K+), intracellular ATP produced a concentration-dependent inhibition of channel activity, with a half-maximal inhibition (Ki) of 2.32 mM. A non-hydrolysable analogue of ATP, AMP(PNP), was similarly effective. In symmetrical K+ (i.e. no extracellular sodium), channel activity was tenfold more sensitive to ATP (Ki of 0.21 mM). A parallel study on ATP-K+ channels from an insulin-secreting beta-cell line (CRI-G1) showed that, in contrast to the neuronal data, extracellular cations had no effect on the ATP sensitivity of the channel.

Adenosine Triphosphate↗