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PubMed1993

Dual contractile effects of ATP released by field stimulation revealed by effects of alpha,beta-methylene ATP and suramin in rat tail artery.

1. The field stimulation-induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2-purinceptor antagonist, suramin, on these parameters were compared with those of alpha,beta-methylene ATP, a P2X-purinoceptor desensitizing agent. 2. Neither alpha,beta-methylene ATP (10 microM) nor suramin (100-500 microM) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP-induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3. Combined blockade of both postjunctional alpha 1- and alpha 2-adrenoceptors by prazosin and yohimbine (both 0.1 microM) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by alpha,beta-methylene ATP (10 microM) and inhibited by suramin in a concentration-dependent manner (10-500 microM; IC50 75 microM) and was hence probably caused by ATP or a related nucleotide. 4. When added first, alpha,beta-methylene ATP (10 microM) or suramin (100-500 microM) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 microM). 5. The K+ channel blocker, tetraethylammonium (10 mM), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic. Addition of alpha,beta-methylene ATP (10 microM)or suramin (100-500 microM) abolished the large initial twitch component of this contraction and depressed the tonic phase.6. Like alpha,beta-methylene ATP, suramin (500 microM) had no effect on the contraction caused by exogenous NA (1O nM-l10 microM) or KCI (60 mM); both agents almost abolished the contraction caused by ATP(100 microM).7. In conclusion, (i) the contractile response of rat tail artery to electrical field stimulation is mediated by both ATP and NA, and is thus an expression of ATP-NA co-transmission, (ii) the released ATP exerts two opposite effects via 'P2x-like' purinoceptors, triggering the initial rapid phase of the neurogenic contraction and restricting the NA-mediated component of the contraction; and (iii) the source and possible physiological role of the ATP which causes the inhibitory effect are unknown at present.

Adenosine Triphosphate↗

PubMed2001

Phospholipids as modulators of K(ATP) channels: distinct mechanisms for control of sensitivity to sulphonylureas, K(+) channel openers, and ATP.

Recent work has established membrane phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP(2)) as potent regulators of K(ATP) channels controlling open probability and ATP sensitivity. We here investigated the effects of phospholipids on the pharmacological properties of cardiac type K(ATP) (Kir6.2/SUR2A) channels. In excised membrane patches K(ATP) channels showed considerable variability in sensitivity to glibenclamide and ATP. Application of the phosphatidylinositol phosphates (PIPs) phosphatidylinositiol-4-phosphate, PIP(2), and phosphatidylinositol-3,4,5-trisphosphate reduced sensitivity to ATP and glibenclamide closely resembling the native variability. Insertion of the patch back into the oocyte (patch-cramming) restored high ATP and glibenclamide sensitivity, indicating reversible modulation of K(ATP) channels via endogenous PIPs-degrading enzymes. Thus, the observed variability seemed to result from differences in the membrane phospholipid content. PIP(2) also diminished activation of K(ATP) channels by the K(+) channel openers (KCOs) cromakalim and P1075. The properties mediated by the sulphonylurea receptor (sensitivity to sulfonylureas and KCOs) seemed to be modulated by PIPs via a different mechanism than ATP inhibition mediated by the Kir6.2 subunits. First, polycations abolished the effect of PIP(2) on ATP inhibition consistent with an electrostatic mechanism but only weakly affected glibenclamide inhibition and activation by KCOs. Second, PIP(2) had clearly distinct effects on the concentration-response curves for ATP and glibenclamide. However, PIPs seemed to mediate the different effects via the Kir6.2 subunits because a mutation in Kir6.2 (R176A) attenuated simultaneously the effects of PIP(2) on ATP and glibenclamide inhibition. Finally, experiments with various lipids revealed structural features necessary to modulate K(ATP) channel properties and an artificial lipid (dioleoylglycerol-succinyl-nitriloacetic acid) that mimicked the effects of PIPs on K(ATP) channels.

Adenosine Triphosphate↗

PubMed1997

Proteolytic activity of the ATP-dependent protease HslVU can be uncoupled from ATP hydrolysis.

HslVU is a new Escherichia coli ATP-dependent protease composed of two multimeric complexes: the HslU ATPase and the HslV peptidase. Prior studies indicated that HslVU requires ATP hydrolysis for the cleavage of peptides and proteins. We show here that ATP concentrations that activate hydrolysis of benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin are 50-100 fold lower than those necessary for degradation of proteins (e.g. casein). Also, the nonhydrolyzable analogs of ATP, 5'-adenylyl beta, gamma-imidodiphosphate (AMP-PNP) and adenosine 5'-(alpha, beta-methylene)triphosphate, can support peptide hydrolysis, but only after an initial time lag not seen with ATP. This delay decreased at higher temperatures and with higher HslU or HslV concentrations and was eliminated by preincubation of HslU and HslV together. Thus, ATP hydrolysis accelerates the association of HslU and HslV, which occurs slowly with the nonhydrolyzable analog. The addition of KCl stimulated 4-6-fold the peptidase activity with AMP-PNP present and eliminated the time lag, but KCl had no stimulatory effect with ATP. NH4+ and Cs+ had similar effects as K+, but Na+ and Li+ were ineffective. AMP-PNP by itself supported hydrolysis of casein and other polypeptides only 20% as well as ATP, but in the presence of K+, Cs+, or NH4+, AMP-PNP activated casein degradation even better than ATP, although it was not hydrolyzed. In addition, MgCl2, MnCl2, and CaCl2 allowed some peptidase and caseinase activity in the absence of any nucleotide. However, Mn2+ and Ca2+, unlike Mg2+, abolished ATP hydrolysis and prevented further activation by ATP or AMP-PNP. These findings indicate that ATP binding to a high affinity site triggers the formation of an active state capable of peptide cleavage, although ATP hydrolysis facilitates this process. Rapid degradation of proteins requires a distinct state of the enzyme, which is normally reached through ATP hydrolysis at low affinity sites. However, AMP-PNP binding together with K+ can induce a form of HslVU that degrades proteins without energy consumption.

ATP-Dependent Proteases↗

PubMed2004

Mutation in nucleotide-binding domains of sulfonylurea receptor 2 evokes Na-ATP-dependent activation of ATP-sensitive K+ channels: implication for dimerization of nucleotide-binding domains to induce channel opening.

The ATP-sensitive K+ (KATP) channel is composed of a sulfonylurea receptor (SUR) and a pore-forming subunit, Kir6.2. SUR is an ATP-binding cassette (ABC) protein with two nucleotide-binding domains (NBD1 and NBD2). Intracellular ATP inhibits KATP channels through Kir6.2 and activates them through NBDs. However, it is still unknown how ATP-bound NBDs activate KATP channels. A prokaryotic ABC protein, MJ0796, which is entirely NBD, forms a dimer in the presence of Na-ATP when its glutamate at position 171 is substituted with glutamine. Mg2+ or K+ destabilizes the dimer. We made the corresponding mutation in the NBD1 (D834N) and/or NBD2 (E1471Q) of SUR2A and SUR2B. As measured in the inside-out configuration of the patch-clamp method, SUR2x(D834N, E1471)/Kir6.2 channels mediated significantly larger currents in the presence of internal 1 mM Na-ATP than K-ATP alone or Mg-ATP. The response to Na-ATP resulted from an increase in the open probability but not single-channel amplitude of the channels and was abolished by glibenclamide (10(-5) M). In the presence of 1 mM Mg2+ -free ATP, Na+ increased the activity of the channels in a concentration-dependent manner. The Na-ATP-dependent activation was never observed with KATP channels including either the wild-type SUR2x, SUR2x(D834N), or SUR2x(E1471). Nicorandil activated SUR2x(D834N, E1471Q)/Kir6.2 channels more strongly in the presence of Na-ATP than K-ATP alone, whereas the reverse was true for wild-type SUR2x/Kir6.2 channels. Therefore, it is likely that NBDs of SUR2x dimerize in response to ATP and nicorandil. The dimerization induces the opening of the KATP channel, probably by causing a conformational change of SUR2x.

ATP-Binding Cassette Transporters↗

PubMed1983

Dependence of ATP-citrate lyase kinase activity on the phosphorylation of ATP-citrate lyase by cyclic AMP-dependent protein kinase.

ATP-citrate lyase from rat liver and adipose tissue is phosphorylated by either ATP-citrate lyase kinase or catalytic subunit of cyclic AMP-dependent protein kinase to 0.5-0.6 mol/subunit. We previously demonstrated that the site phosphorylated by ATP-citrate lyase kinase (peptide B) is different from that phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase (peptide A) (Ramakrishna, S., Pucci, D. L., and Benjamin, W.B. (1981) J. Biol. Chem. 256, 10213-10216). ATP-citrate lyase phosphorylation by both protein kinases added simultaneously was increased synergistically. When ATP-citrate lyase was first phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase, the net phosphorylation of the fragments subsequently phosphorylated by lyase kinase increased about 6-fold. However, when ATP-citrate lyase was first phosphorylated by lyase kinase, there was no effect on the subsequent phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Alkaline phosphatase-dephosphorylated ATP-citrate lyase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase to 0.9-1.0 mol/subunit. However, dephospho-ATP-citrate lyase was not phosphorylated by lyase kinase. The addition of both protein kinases simultaneously phosphorylated ATP-citrate lyase up to 2 mol/subunit. Phosphorylation of dephospho-ATP-citrate lyase first by catalytic subunit of cyclic AMP-dependent protein kinase and ATP enabled the lyase to be phosphorylated by lyase kinase. Peptide mapping and phosphoamino acid analysis of dephospho-ATP-citrate lyase phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and/or lyase kinase conclusively showed that phosphorylation of ATP-citrate lyase by ATP-citrate lyase kinase was completely dependent on peptide A phosphorylation by cyclic AMP-dependent protein kinase. Furthermore, increased phosphorylation when both protein kinases were added simultaneously was due to increased phosphorylation at peptide B.

ATP Citrate (pro-S)-Lyase↗

PubMed1980

ATP-induced lysis of rat parotid secretory granules: possible role of ATP in exocytotic release.

Secretory vesicles isolated from a variety of mammalian tissues are known to lyse and thereby release their secretory products when exposed to ATP. This process, which will be termed ATP-induced lysis, has been studied most extensively using adrenal chromaffin-granule preparations. We report here that ATP causes the lysis of a highly purified preparation of rat parotid secretory granules. The rate of granule lysis was measured spectrophotometrically, and ATP-induced lysis was expressed as the increase in the rate of lysis (r = % lysis per min) when ATP was added. This lytic process was characterized with respect to pH, temperature, osmolarity, and the ionic composition of the media. ATP-induced lysis of parotid granules was found to have the following properties in common with the extensively characterized chromaffin-granule process: 1. It is a saturable function of ATP with half-maximal rates observed at 0.5 +/- 0.1 mM ATP. 2. It is temperature dependent, eg, r = 6.1 +/- 2.1%/min at 30 degrees C vs 12.2 +/- 2.5%/min at 37 degrees C. 3. It is inhibited in hyperosmotic media, eg, r = 5.3 +/- 0.3%/min at 0.3 OsM vs 0.8 +/- 0.2%/min at 0.4 OsM. 4. It shows a nucleotide preference of ATP = GTP greater than ADP greater than AMP greater than CTP = ITP. 5. It has an anion requirement. The above findings, combined with reports of ATP-induced lysis of cholinergic, insulin, and posterior-pituitary vesicles, imply that ATP-induced lysis may reflect an ATP-dependent property of all secretory vesicles, and as such, this vesicle property could play a similar role in each exocytotic release process. Using a model system, Miller and Racker [22] made a surprising finding that the extent of which liposomes fuse with a black lipid membrane depends on the osmotic gradient across the vesicle membrane. In view of the osmotic dependence of ATP-induced lysis in this and other secretory-vesicle preparations, we postulate that ATP may prime secretory vesicles for fusion with the plasma membrane by inducing and/or maintaining an osmotic gradient across the vesicle membrane.

Adenosine Triphosphate↗

PubMed1999

Regulation of intracellular ATP concentration under conditions of reduced ATP consumption in pancreatic islets.

ATP is the most important factor in glucose-induced insulin secretion in pancreatic beta-cells, but examination of intracellular differences in ATP concentration is difficult because ATP production and consumption occur simultaneously. In the present study, we measured the ATP concentration under the condition of a reduced ATP requirement by omitting extracellular Ca(2+) and inhibiting Na-K ATPase. The ATP concentration in islets incubated with 16.7 mM glucose in the absence of Ca(2+) for 30 min was increased by about 1. 9-fold more than in the presence of Ca(2+). The increment was extracellular Ca(2+)-dependent, and was completely abolished by the metabolic inhibitors dinitrophenol and iodoacetic acid. The Ca channel blockers including nitrendipine and Ni(2+) did not affect the ATP concentration in islets incubated with 16.7 mM glucose in the presence of Ca(2+). However, when thapsigargin and suramin, inhibitors of Ca-ATPase at the endoplasmic reticulum, were added to Ca channel blockers in the presence of ambient Ca(2+), the intraislet ATP content was increased, similarly to that under Ca-free conditions. But thapsigargin did not further augment the ATP concentration in the islet with 16.7 mM glucose in the absence of Ca(2+). On the other hand, the suppression of Na-K ATPase by ouabain rather reduced the ATP concentration augmented by omission of extracellular Ca(2+). In addition, vanadate, a blocker of Ca-ATPase at the plasma membrane, failed to increase the ATP concentration in the islets. These data suggest that the increment of ATP concentration in the absence of Ca(2+) is attributable to the reduced ATP requirement due to stopping of the Ca-ATPase activity at the endoplasmic reticulum, and that the intracellular ATP concentration is differently regulated by Na-K ATPase at plasma membrane and by Ca-ATPase at endoplasmic reticulum.

Adenosine Triphosphate↗

PubMed1986

ATP maintains ATP-inhibited K+ channels in an operational state.

In patch-clamp records of K+ ATP channels in an insulin-secreting cell line (RINm5F) inhibition evoked by exposing the internal surface of the membrane to ATP is followed not just by the recovery of K+ ATP channel activity when the ATP is removed but by a marked activation of K+ ATP channels. This phenomenon is not a direct consequence of channel closure as inhibition induced by quinidine and quinine is followed upon the removal of the drug only by the recovery of K+ ATP channel activity and not by post-inhibitory activation. If ATP is applied to the exposed internal surface of a membrane patch when all of its K+ ATP channel have run down subsequent removal of the ATP causes their activation. The magnitude and duration of the reactivation of K+ ATP channels is shown to depend upon both the concentration of ATP and the length of time for which the membrane is exposed to ATP. We therefore have a paradoxical situation in that K+ channels which are inhibited by intracellular ATP require intracellular ATP to retain the ability to open.

Adenosine Triphosphate↗

PubMed1996

ATP and beta,gamma-methylene ATP produce relaxation of guinea-pig isolated trachealis muscle via actions at P1 purinoceptors.

Adenosine 5'-triphosphate (ATP), beta, gamma-methylene ATP and alpha, beta-methylene ATP produced relaxation of carbachol-precontracted isolated trachealis muscle from the guinea-pig in the presence of indomethacin (2.8 microM) and the adenosine uptake inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI; 300 nM). The potency order for ATP and analogues was: beta, gamma-methylene ATP = ATP > alpha, beta-methylene ATP = uridine 5'-triphosphate (UTP) = 2-methylthio ATP. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) also caused relaxation. Relaxations to ATP, beta, gamma-methylene ATP, adenosine and NECA were not inhibited by the P2 purinoceptor antagonist suramin (100 microM), but were inhibited by the P1 purinoceptor antagonist 8-sulphophenyltheophylline (140 microM). NBTI significantly potentiated adenosine and ATP but not beta, gamma-methylene ATP or NECA. The data are compatible with the idea that beta, gamma-methylene ATP could interact directly with P1 purinoceptors while ATP acts indirectly at P1 purinoceptors via conversion to adenosine.

Adenosine Triphosphate↗

PubMed1994

Influence of ATP and ATP agonists on the physiology of the isolated semicircular canal of the frog (Rana pipiens).

In the present study, the influence of extracellular ATP and ATP agonists in the physiology of the vestibular organs was examined, using the in vitro model of the isolated semicircular canal of the frog (Rana pipiens). The firing activity of the afferent nerve, the d.c. nerve potential and the transepithelial potential were measured in the absence and presence of mechanical stimulation of the sensory epithelium. Administration of ATP into the perilymphatic compartment, from 10(-12) to 10(-3) M, increased the firing rate of the afferent fibers recorded in the absence of mechanical stimulation. Recordings of the d.c. nerve potential indicated that the afferent fibers were hyperpolarized. The presence of the purine also modified the transepithelial potential. During mechanical stimulation of the sensory epithelium, both the evoked afferent firing and the evoked variation of the d.c. nerve potential were reduced in the presence of ATP. However, ATP did not effect the evoked modulation of the transepithelial potential, evoked by the mechanical stimulation. Administration of the P2x purinoceptor agonists, alpha, beta-methylene-ATP and beta, gamma-methylene-ATP, at concentrations between 10(-12) and 10(-3) M, did not significantly modify the different bioelectrical activities investigated. In contrast, 2-methylthio-ATP, a P2y purinoceptor agonist, more potent and efficacious than ATP in its effect on the spontaneous firing. Concurrently, no modification of the d.c. nerve potential, the transepithelial potential and their variation during mechanical stimulation was observed. In opposition to the ATP effect, the total amplitude of the evoked firing was increased in the presence of 2-methylthio-ATP. These data suggest that extracellular ATP, present in the perilymphatic compartment, may act as a neuromodulator in the vestibular physiology. The effects of the purine appear to be mediated by the activation of a P2y subtype of purinoceptor. The absence of an effect of ATP and 2-methylthio-ATP on the evoked variation of the transepithelial potential suggest that the purine did not affect the processes responsible for the generation of the receptor potential but more likely modified the mechanisms involved in the release of the neurotransmitter from the hair cells and/or acted on the afferent endings.

Action Potentials↗

PubMed2005

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis.

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.

Adenosine Diphosphate↗

PubMed2005

Does conversion of ATP to adenosine terminate ATP-stimulated vasopressin release from hypothalamo-neurohypophyseal explants?

ATP stimulates vasopressin (VP) release from explants of the hypothalamo-neurohypophyseal system (HNS), but the response is not sustained for the duration of exposure to ATP. Since adenosine, a metabolite of ATP, inhibits VP release from neurohypophysial terminals and adenosine receptors (AR) are expressed in supraoptic nucleus (SON) neurons, we postulated that conversion of ATP to adenosine contributed to termination of ATP-stimulated VP release from HNS explants. This was tested using a non-selective AR antagonist, 5-amino-9-chloro-2-(2-furyl)-1, 2, 4-triazolo [1, 5-c] quinazoline (CGS-15943). CGS-15943 did not affect basal VP release and did not alter the initial response to ATP. A selective A1R antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), increased basal VP release at 1 microM, without altering the response to ATP. However, at a higher concentration of DPCPX (10 microM), VP release was enhanced by ATP for an extended period of time. Inhibition of the enzymatic conversion of ATP to adenosine using a combination of a potent ecto-5'-nucleotidase inhibitor, alpha,beta-methylene adenosine 5'-diphosphate (AMP-CP), and a competitive substrate for ecto-5'-nucleotidase (guanosine monophosphate, GMP) did not affect basal VP release. Enzymatic inhibition did slightly prolong the response to ATP, but it was not sustained for the duration of exposure to ATP. We conclude that an endogenous inhibitory influence of adenosine decreases basal VP release from HNS explants and that conversion of exogenously applied ATP to adenosine contributes to termination of ATP-induced stimulation of VP release, but additional mechanisms such as receptor desensitization also limit the response to extended exposure to ATP.

5'-Nucleotidase↗

PubMed2000

Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis.

The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.

Adenosine Diphosphate↗

PubMed1988

Relaxation of muscle fibers with adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and by laser photolysis of caged ATP[gamma S]: evidence for Ca2+-dependent affinity of rapidly detaching zero-force cross-bridges.

The relationship between the mechanical and biochemical states of the muscle cross-bridge cycle and the control of contraction were investigated by using the nucleotide analogs adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and caged ATP[gamma S] [the O-1(2-nitrophenyl)ethyl P3-ester of ATP[gamma S]]. ATP[gamma S] interacts with actomyosin in a manner similar to ATP but is hydrolyzed (by a factor of 500) more slowly. Generation of ATP[gamma S] by photolysis of caged ATP[gamma S] within a permeabilized fiber in rigor in the absence of Ca2+ relaxed tension and stiffness as occurs with ATP. The transient rise in tension prior to final relaxation observed with photolysis of caged ATP was absent with caged ATP[gamma S]. This result suggests that following detachment of a cross-bridge, ATP is normally hydrolyzed before force generation. In the presence of Ca2+, photolysis of caged ATP[gamma S] within rigor fibers caused tension to relax fully but significant stiffness remained. Stiffness also developed without concomitant tension when Ca2+ concentration was raised from less than 1 nM to 30 microM in the presence of ATP[gamma S]. The amplitude of the tension response to ramp stretches in the presence of Ca2+ and ATP[gamma S] increased with ramp stretch velocity, suggesting that the cross-bridges have detachment rate constants extending into the 10(3) s-1 range. The results provide evidence that the Ca2+-regulatory system can directly control attachment of cross-bridges into states before the power stroke.

Actomyosin↗

PubMed1996

ATP synthase. Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP.

Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.

Adenosine Diphosphate↗

PubMed1998

V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP.

The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 degreesC. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 microM; from this, an apparent Km of 240 +/- 24 microM and a Vmax of 5.2 +/- 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit. These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.

Adenosine Diphosphate↗

PubMed1999

The role of lysine 185 in the kir6.2 subunit of the ATP-sensitive channel in channel inhibition by ATP.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, forms ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on Kir6.2. 2. Previous studies have shown that mutation of the lysine residue at position 185 (K185) in the C-terminus of Kir6.2 to glutamine, decreased the channel sensitivity to ATP without affecting the single-channel conductance or the intrinsic channel kinetics. This mutation also impaired 8-azido[32P]-ATP binding to Kir6.2. 3. To determine if K185 interacts directly with ATP, we made a range of mutations at this position, and examined the effect on the channel ATP sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 4. Substitution of K185 by a positively charged amino acid (arginine) had no substantial effect on the sensitivity of the channel to ATP. Mutation to a negatively charged residue markedly decreased the channel ATP sensitivity: the Ki for ATP inhibition increased from 85 microM to >30 mM when arginine was replaced with aspartic acid. Substitution of neutral residues had intermediate effects. 5. The inhibitory effects of ADP, ITP and GTP were also reduced when K185 was mutated to glutamine or glutamate. 6. The results indicate that a positively charged amino acid at position 185 is required for high-affinity ATP binding to Kir6.2. Our results demonstrate that ATP does not interact with the side-chain of K185. It remains unclear whether ATP interacts with the backbone of this residue, or whether its mutation influences ATP binding allosterically.

Adenosine Triphosphate↗

PubMed1976

ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump: evidence for allosteric effects of intracellular ATP and extracellular sodium.

1. A method has been developed for regenerating [gamma(32)P]ATP of constant specific activity within resealed red cell ghosts, and for measuring its hydrolysis. The method may be used to follow the hydrolysis of ATP at concentrations down to 1 muM, and for periods long enough for the ATP at these very low concentrations to turn over several hundred times.2. Using this method we have been able to show that the ;uncoupled' efflux of Na caused by the Na pump when resealed red cell ghosts are incubated in (Na + K)-free media is associated with a hydrolysis of ATP. The stoicheiometry is roughly 2-3 Na ions expelled per molecule of ATP hydrolysed.3. Measurements of ATP hydrolysis and Na efflux as functions of intracellular ATP concentration have shown that uncoupled Na efflux, and its associated ATP hydrolysis, are saturated at intracellular ATP concentrations in the region of 1 muM.4. Measurement of ATP hydrolysis as a function of ATP concentration in resealed ghosts incubated in a K-containing medium gave a complicated activation curve suggesting the involvement of high-affinity (K(m)ca. 1 muM) and low-affinity (K(m)ca. 100 muM) sites.5. When resealed ghosts containing about 1 muM-ATP were incubated in a Na-free or in a high-Na medium, the addition of K to the medium reduced the rate of ouabain-sensitive ATP hydrolysis.6. Ouabain-sensitive ATP hydrolysis in resealed ghosts incubated in K-free choline media was inhibited by external Na at low concentrations (K(i) < 1 mM), but this inhibition was reversed as the external Na concentration was further increased.7. The results show that uncoupled Na efflux may be thought of as the transport mode associated with Na-ATPase activity, just as Na-K exchange is the transport mode associated with (Na + K)-ATPase activity. The significance of the differences between uncoupled Na efflux and Na-ATPase activity, on the one hand, and Na-K exchange and (Na + K)-ATPase activity, on the other, is discussed.

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