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PubMed1991

Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP.

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

PubMed2001

Pharmacological comparison of native mitochondrial K(ATP) channels with molecularly defined surface K(ATP) channels.

Many mammalian cells have two distinct types of ATP-sensitive potassium (K(ATP)) channels: the classic ones in the surface membrane (sK(ATP)) and others in the mitochondrial inner membrane (mitoK(ATP)). Cardiac mitoK(ATP) channels play a pivotal role in ischemic preconditioning, and thus represent interesting drug targets. Unfortunately, the molecular structure of mitoK(ATP) channels is unknown, in contrast to sK(ATP) channels, which are composed of a pore-forming subunit (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). As a means of probing the molecular makeup of mitoK(ATP) channels, we compared the pharmacology of native cardiac mitoK(ATP) channels with that of molecularly defined sK(ATP) channels expressed heterologously in human embryonic kidney 293 cells. Using mitochondrial oxidation to index mitoK(ATP) channel activity in rabbit ventricular myocytes, we found that pinacidil and diazoxide open mitoK(ATP) channels, but P-1075 does not. On the other hand, 5-hydroxydecanoic acid (5HD), but not HMR-1098, blocks mitoK(ATP) channels. Although pinacidil is a nonselective activator of expressed sK(ATP) channels, diazoxide did not open channels formed by Kir6.1/SUR2A, Kir6.2/SUR2A (known components of cardiac sK(ATP) channels) or Kir6.2/SUR2B. P-1075 activated all the K(ATP) channels, except Kir6.1/SUR1 channels. Glybenclamide potently blocked all sK(ATP) channels, but 5HD only blocked channels formed by SUR1/Kir6.1 or Kir6.2 (IC(50)s of 66 and 81 microM, respectively). This potency is similar to that for block of mitoK(ATP) channels (IC(50) = 95 microM). In addition, HMR-1098 potently blocked Kir6.2/SUR2A channels (IC(50) = 1.5 microM), but was 67 times less potent in blocking Kir6.1/SUR1 channels (IC(50) = 100 microM). Our results demonstrate that mitoK(ATP) channels closely resemble Kir6.1/SUR1 sK(ATP) channels in their pharmacological profiles.

ATP-Binding Cassette Transporters↗

PubMed1990

Inhibition of recA protein promoted ATP hydrolysis. 1. ATP gamma S and ADP are antagonistic inhibitors.

ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms. In both cases, at least two modes of inhibition are observed. For ADP, the first mode is competitive inhibition. The second mode is manifested by dissociation of recA protein from DNA. These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations. Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction. The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio. In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations. At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor. At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis. These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) [poly(dT)] cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used. ATP gamma S can also relieve the inhibitory effect of ADP under some conditions. ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive. The ATP gamma S (ATP) conformation is active in ATP hydrolysis. The ADP conformation is inactive.

Adenosine Diphosphate↗

PubMed2004

ATP-induced ATP release from astrocytes.

Propagation of interastrocyte Ca2+ waves is mediated by diffusion of extracellular adenosine triphosphate (ATP), and may require regenerative release of ATP. The ability of ATP to initiate release of intracellular ATP was assessed by labeling adenine nucleotide pools in astrocyte cultures with 14C-adenine. The 14C-purines released during exposure to ATP were then identified by thin-layer chromatography. ATP treatment caused a five-fold increase in release of 14C-ATP but not 14C-ADP or 14C-AMP, indicating selectivity for release of ATP. Other P2 receptor agonists also caused significant 14C-ATP release, and the P2 receptor antagonists suramin, reactive blue-2 and pyridoxalphosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) inhibited ATP-induced 14C-ATP release to varying degrees, suggesting the involvement of a P2 receptor. ATP-induced 14C-ATP release was not affected by chelation of intracellular Ca2+ with BAPTA-AM, or by blockers of Ca2+ release from intracellular stores or of extracellular Ca2+ influx, suggesting a Ca2+-independent response. ATP-induced 14C-ATP release was significantly inhibited by non-selective anion channel blockers but not by blockers of ATP-binding cassette proteins, gap junction hemichannels, or vesicular exocytosis. Release of adenine nucleotides induced by 0 Ca2+ was, in contrast, not selective for ATP, and was susceptible to inhibition by gap junction blockers. These findings indicate that astrocytes are capable of ATP-induced ATP release and support a role for regenerative ATP release in glial Ca2+ wave propagation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Acceleration of unisite catalysis of mitochondrial F1-adenosinetriphosphatase by ATP, ADP and pyrophosphate--hydrolysis and release of the previously bound [gamma-32P]ATP.

The effect of ATP, ADP and pyrophosphate (PPi) on hydrolysis and release of [gamma-32P]ATP bound to the high-affinity catalytic site of soluble F1 from bovine heart mitochondria under unisite conditions [Grubmeyer, C., Cross, R. L. & Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100] was studied. In accord with the previous data, it was observed that millimolar concentrations of ATP or ADP added to F1 undergoing unisite hydrolysis of [gamma-32P]ATP accelerated its hydrolysis. PPi also produced a hydrolytic burst of a fraction of the previously bound [gamma-32P]ATP; kinetic data suggested that for production of optimal hydrolysis by PPi of the bound [gamma-32P]ATP, two binding sites with apparent Kd of 27 microM and 240 microM must be filled. The extent of the hydrolytic burst induced by MgPPi was lower than that induced by ADP and ATP. In F1 in which PPi had produced a hydrolytic burst of the bound [gamma-32P]ATP, the addition of ATP induced a second burst of hydrolysis. By filtration experiments and enzyme trapping, it was also studied whether ATP, ADP and PPi produce release of the tightly bound [gamma-32P]ATP. At millimolar concentrations, ATP and ADP brought about release of about 25% of the previously bound [gamma-32P]ATP. At micromolar concentrations, ADP accelerated the hydrolysis of the previously bound [gamma-32P]ATP but not its release. Hence, the hydrolytic and release reactions could be separated, indicating that the two reactions require the occupancy of different sites in F1. With PPi, no release of the tightly bound [gamma-32P]ATP was observed. The ADP induced hydrolysis and release of the F1-bound [gamma-32P]ATP were inhibited by sodium azide to the same extent (60%). Since release of ATP from a high-affinity catalytic site of F1 represents the terminal step of oxidative phosphorylation, the data illustrate that the binding energy of substrates to F1 is critical to the ejection of ATP into the media. The failure of PPi to induce release of [gamma-32P]ATP bound to F1 under unisite conditions is probably due to its lower binding energy.

Adenosine Diphosphate↗

PubMed1997

Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP.

The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of [3H]acetylcholine from the isolated superior cervical ganglion of the rat loaded with [3H]choline were studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a [Ca2+]o-dependent manner. The Na+ channel blocker, tetrodotoxin (1 microM) inhibited the stimulation-evoked release of endogenous ATP and of [3H]acetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10 Hz. when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of [3H]choline was significantly reduced but not inhibited. Hexamethonium, (100 microM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A1-adenosine receptors. When the temperature was reduced to 7 degrees C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9 +/- 5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10 days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the Km (apparent Michaelis constant) and Vmax (apparent maximal velocity) were 475 +/- 24 microM and 3.50 +/- 0.18 nmol/min per mg for ectoATPase and 1550 +/- 120 microM and 14.5 +/- 0.9 nmol/min per mg tissue for 5'-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a [Ca2+]o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue. The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.

Adenosine Triphosphate↗

PubMed2001

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Adenine Nucleotides↗

PubMed1993

The effects of ATP and alpha,beta-methylene-ATP on cytosolic Ca2+ level and force in rat isolated aorta.

1. The effects of a non-selective P2-receptor agonist ATP and a selective P2x-receptor agonist alpha,beta-methylene-ATP on intracellular free Ca2+ level ([Ca2+]i) and force were examined in rat isolated aorta without endothelium. 2. Both ATP (1-1000 microM) and alpha,beta-methylene-ATP (0.1-100 microM) induced transient increase followed by small sustained increase in [Ca2+]i in a concentration-dependent manner. Compared with the force induced by a high concentration of KCl, the force induced by alpha,beta-methylene-ATP was smaller and that induced by ATP was much smaller at a given [Ca2+]i. 3. An L-type Ca2+ channel blocker, verapamil (10 microM), completely inhibited the high K(+)-stimulated [Ca2+]i and force. Verapamil partially inhibited the transient and sustained increases in [Ca2+]i induced by 10 microM alpha,beta-methylene-ATP and the sustained increase but not the transient increase induced by 1 mM ATP. 4. In the absence of extracellular Ca2+ (with 0.5 mM EGTA) 1 mM ATP caused transient increase in [Ca2+]i while 10 microM alpha,beta-methylene-ATP was ineffective 5. ATP, but not alpha,beta-methylene-ATP, increased the tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) level. 6. These data suggest that ATP and alpha,beta-methylene-ATP increase [Ca2+]i by an activation of both L-type and non-L-type Ca2+ channels. In addition, ATP, but not alpha,beta-methylene-ATP, increases [Ca2+]i by a release of Ca2+ from an intracellular Ca2+ store. Possible reasons are discussed as to why the increase in [Ca2+]i due to ATP and alpha,beta-methylene-ATP resulted in only a small contraction.

Adenosine Triphosphate↗

PubMed1982

Source of rapidly labeled ATP tightly to non-catalytic sites on the chloroplast ATP synthetase.

Bound [32P]ATP is found on deenergized, washed chloroplast thylakoids which were illuminated in the presence of ADP and [32P]Pi. Tight binding of [32P]ATP occurred both during and after energization. Different classes of bound [32P]ATP were distinguished on the basis of their rates of formation, susceptibility to hexokinase and displacement by unlabeled ATP. 1. The rates of formation and discharge of the rapidly labeled tightly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation. 2. We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation. 3. Since the rapidly labeled tightly bound [32P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non-catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.

ATP Synthetase Complexes↗

PubMed2001

ATP interaction with the open state of the K(ATP) channel.

The mechanism of ATP-sensitive potassium (K(ATP)) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP2) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact with the open state of the channel is presented here. The mutant Kir6.2[L164C] is very sensitive to Cd2+ block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP. However, 1 mM ATP fully protects the channel from Cd2+ block. Allosteric kinetic models in which the channel can be in either open or closed states with or without ATP bound are considered. Such models predict a pedestal in the ATP inhibition, i.e., a maximal amount of inhibition at saturating ATP concentrations. This pedestal is predicted to occur at >50 mM ATP in the L164C mutant, but at >1 mM in the double mutant L164C/R176A. As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of approximately 40%, with a clear plateau beyond 2 mM. These results indicate that ATP acts as an allosteric ligand, interacting with both open and closed states of the channel.

Adenosine Triphosphate↗

PubMed1992

Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.

Adenosine Triphosphate↗

PubMed1990

Existence of a low-affinity ATP-binding site in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles: evidence from binding of 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP and -[3H]ATP.

ATP-binding sites in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles were titrated with 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP (TNP-AMP) or -[3H]ATP (TNP-ATP) in the absence of Ca2+ at pH 7.0 and 0 degrees C by using a centrifugation procedure. In some measurements, the bound TNP-nucleotides were chased with ATP. The data were analyzed by best-fit computer programs as well as by Scatchard plots. The results showed the existence of 1 mol of TNP-AMP binding sites with high affinity (Kd = 7.62 nM) per mole of phosphorylatable sites. The affinity of these sites for ATP (Kd = 10.1 microM) agreed with that of catalytic sites for ATP in the absence of Ca2+. The results further showed the existence of 2 mol of TNP-ATP binding sites with uniform affinity (Kd = 156 nM) per mole of phosphorylatable sites. Half of the bound TNP-ATP was fully chased by low concentrations of ATP. The affinity of this class of the sites for ATP (Kd = 8.9 microM) again agreed with that of catalytic sites for ATP. The other half of the bound TNP-ATP was fully chased only by much higher concentrations of ATP. Thus, the affinity of this class of the sites for ATP (Kd = 791 microM) was much lower than that of catalytic sites for ATP. Similar measurements were performed with sarcoplasmic reticulum vesicles pretreated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Although the affinities for TNP-ATP and for ATP were appreciably altered by this pretreatment, the results were essentially the same as those obtained with native vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1998

ATP synthesis by F0F1-ATP synthase independent of noncatalytic nucleotide binding sites and insensitive to azide inhibition.

ATP hydrolyzing activity of a mutant alpha3beta3gamma subcomplex of F0F1-ATP synthase (DeltaNC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E. , Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221). This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F0F1-ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, DeltaNC F0F1-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by DeltaNC F0F1-ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by DeltaNC F0F1-ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.

Adenosine Triphosphate↗

PubMed1996

The binding characteristics of a human bladder recombinant P2X purinoceptor, labelled with [3H]-alpha beta meATP, [35S]-ATP gamma S or [33P]-ATP.

1. The binding of [3H]-alpha beta meATP, [35s]-ATP gamma S and [alpha 33P]-ATP to a human bladder P2X purinoceptor, transiently expressed in CHO-K1 cells using the Semliki Forest Virus (SFV) expression system, was examined. The characteristics of the binding sites were compared with results obtained in rat vas deferens, a tissue in which the radioligands are thought to label P2X purinoceptors and in which the endogenous P2X purinoceptor displays high homology with the human bladder P2X purinoceptor. 2. In non-infected CHO-K1 cells, 100 microM ATP evoked only small inward currents (40 pA) in approximately 30% of the cells when studied by the whole-cell voltage clamp technique. In membranes prepared from either these non-infected cells or cells infected with SFV containing the LacZ gene (SFV-LacZ), [3H]-alpha beta meATP bound with low affinity (pKd = 7.04; Bmax = 8.88 pmol ml-1 protein) and there was only a low density of [35S]-ATP gamma S binding sites (pKd = 8.74; Bmax = 358 fmol ml-1 protein). These binding sites differed from those present in rat vas deferens. Thus, pIC50 values for alpha beta meATP (6.5) and L-beta gamma meATP (4.0) at the [3H]-alpha beta meATP binding sites in non-infected CHO-K1 cells were much lower than the respective pIC50 values of 8.3 and 7.7, determined in rat vas deferens. Similarly, affinity estimates (pIC50 values) for ATP (6.82), 2-meS-ATP (5.43), ATP gamma S (7.06) and alpha beta meATP (4.84) at the [35S]-ATP gamma S binding sites in non-infected CHO-K1 cells were up to 2291 fold lower than the respective values of 9.01, 8.79, 8.73 and 7.57, determined in rat vas deferens. 3. In CHO-K1 cells infected using SFV containing the cDNA for the human bladder P2X purinoceptor (SFV-h.P2X), ATP, 2-meS-ATP and alpha beta meATP evoked large inward currents (2-7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV-h.P2X infected cells, [3H]-alpha beta meATP binding was increased, compared to that measured in the non infected or SFV-LacZ infected cells, with only high affinity [3H]-alpha beta meATP binding sites being detected (pKd = 9.21; Bmax = 3.54 pmol mg-1 protein). The pIC50 values for alpha beta meATP (8.2) and L-beta gamma meATP (7.2) in competing for these sites were the same or similar to the values determined in rat vas deferens. 4. A high density of [35H]-ATP gamma S binding sites (pKd = 9.09; Bmax = 6.82 pmol mg-1 protein) was also present in the membranes from CHO-K1 cells infected with SFV-h.P2X and affinity estimates (pIC50 values) for ATP (8.93), 2-meS-ATP (8.23), ATP gamma S (8.08), and alpha beta meATP (7.17) at competing for these sites were as much as 631 fold higher than the respective values determined in non-infected CHO-K1 cells but were close to the values determined in rat vas deferens. Similar data were obtained with [alpha 33P]-ATP as radioligand. 5. These data suggest that [3H]-alpha beta meATP, [35S]-ATP gamma S and [33P]-ATP label the human bladder recombinant P2X purinoceptor expressed in CHO-K1 cells following infection with SFV-h.P2X and provide further corroborative evidence to support the contention that the high affinity binding sites for these radioligands in rat vas deferens are P2X purinoceptors.

Adenosine Triphosphate↗

PubMed1996

Proton potentiation of ATP-gated ion channel responses to ATP and Zn2+ in rat nodose ganglion neurons.

1. The modulation by protons of ATP-gated ion channel responses to ATP and Zn2+ was studied in freshly isolated rat nodose ganglion neurons using the whole cell patch-clamp technique. 2. Reduced external pH enhanced, whereas elevated external pH suppressed, current activated by 10 microM ATP. The pH producing the half-maximal effect (EC50) at this ATP concentration was 7.1. 3. Acidification shifted the ATP concentration-response curve to the left, decreasing the EC50 for ATP, and alkalinization shifted the ATP concentration-response curve to the right, increasing the EC50 for ATP. Fitting the data to a single-site pH model yielded an apparent pKa of the site on the ATP-gated ion channel of 7.6. Between pH 6.8 and 7.8, a change of 0.1 pH unit was calculated to change the ATP EC50 by 4.03 microM. Changing pH did not alter the maximal response to ATP. 4. The potentiating effect of protons appeared to be due to a direct action on the ATP-gated channel, as it could not be explained by an increase in the concentration of one or more species of ATP. 5. Lowering pH also increased the potency of Zn2+ for enhancement of ATP-activated current without altering its maximal response. Changing the pH from 7.3 to 6.8 changed the Zn2+ EC50 from 12 to 1.7 microM. 6. The potentiation of ATP-activated current by protons could not be attributed solely to an increase in the affinity of the receptor for Zn2+, as the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine did not alter the effect of protons. 7. Protons and Zn2+ do not appear to act at the same site on ATP-gated channels, as responses to maximally effective concentrations of Zn2+ were enhanced further by protons and vice versa. 8. These results suggest that protons regulate the function of P2X purinoceptors in rat nodose ganglion neurons by modulating the affinity of the binding sites for ATP and Zn2+ on these receptor channels.

Adenosine Triphosphate↗

PubMed1994

Photochemical labeling and inhibition of Na,K-ATPase by 2-Azido-ATP. Identification of an amino acid located within the ATP binding site.

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.

Adenosine Triphosphate↗

PubMed1994

Relationships between cytosolic [ATP], [ATP]/[ADP] and ionic fluxes in the perfused rat heart: A 31P, 23Na and 87Rb NMR study.

In order to assess the relationship between cytosolic [ATP] or [ATP]/[ADP] and the intracellular Na+ concentration ([Na+]i), we have used the phosphate trap 2-deoxy-D-glucose (DG) to alter the high energy phosphate levels in rat cardiomyocytes. Pyruvate-perfused rat hearts were treated with 2 mM DG in the presence of 10IU/l of insulin for 28 min, followed by perfusion with DG without insulin for 60 min. The DG + insulin treatment resulted in dramatic changes in the 31P NMR spectra: phosphocreatine (PCr) and total ATP decreased (to 15 and 35%, respectively) and deoxyglucose-6-phosphate accumulated, with little change in either inorganic phosphate or intracellular pH. These changes corresponded to a decrease in cytoplasmic [ATP] (from 7.6 to 1.8 mM), [ATP]/[ADP] (from 494 to 24) and ATP affinity [A(ATP), by 8.9 kJ/mol] and an increase in [ADP] (five-fold) and free [Mg2+] (two-fold). Subsequent perfusion with DG--insulin resulted in slow recovery of PCr, [ATP]/[ADP] and A(ATP) such that the "low energy" state lasted an additional 16 min during which ATP remained low and constant. There were no detectable changes in the intracellular Na+ content as assessed by shift reagent-aided 23Na NMR at the end of DG + insulin treatment (98 +/- 18%, 28-36 min of the protocol). In addition, there was no change in the Rb+ influx rate as measured by 87Rb NMR at the beginning of insulin washout which was achieved by replacing 20% of the KCl with RbCl ([K+] = 3.76 mM, [Rb+] = 0.94 mM). During DG + insulin treatment the pressure-rate product (PRP) decreased by half and was restored upon insulin washout to 80% of its initial value both in the presence and in the absence of the shift reagent [5 mM Dy (triethylenetetraminehexaacetate)3-]. These data imply that unfavorable thermodynamic [low A(ATP)] and kinetic (low [ATP] and [ATP]/[ADP]) conditions induced by DG treatment do not inhibit Na+, K(+)-ATPase activity. We speculate that during anoxia when changes in [ATP]/[ADP] are comparable to those induced by DG treatment, the observed increase in [Na+]i is not due to inhibition of the Na+ pump by reduced [ATP] or [ATP]/[ADP].

Adenosine Diphosphate↗

PubMed1983

A time lag in the onset of ATP-Pi exchange catalyzed by purified ATP-synthase (CF0-CF1) proteoliposomes and by chloroplasts.

The time course of ATP-Pi exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made. (i) The onset of 32Pi incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 degrees C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate. (ii) The initial lag is independent of Mg-ATP concentration in the range 0.2-5 mM and of the presence of ADP. In contrast, the steady-state rate of ATP-Pi exchange has an apparent Km of 0.3 mM for Mg-ATP and is stimulated by ADP. (iii) Increasing the temperature from 30 to 45 degrees C decreases the lag from 6 min to zero. The steady-state rate of ATP-Pi exchange is affected to a much smaller extent by the temperature in this range. (iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate. (v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system. (vi) Light-triggered ATP-Pi exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP-Pi exchange does not reflect the time required for the buildup of a protomotive force (delta - mu H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an "exclusive ATPase state" to a "reversible ATP-synthase state". This slow transition is not directly coupled to a trans-membrane pH or potential gradient.

Adenosine Diphosphate↗