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PubMed2002

Replacement of several single amino acid side chains exposed to the inside of the ATP-binding pocket induces different extents of affinity change in the high and low affinity ATP-binding sites of rat Na/K-ATPase.

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na(+)/K(+)-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na(+)/K(+)-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na(+)/K(+)-ATPase or low affinity ATP inhibition of K(+)-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K(0.5). However, both the affinities for the K480E were reduced to approximately 1/20. The turnover number of the Na(+)/K(+)-ATPase and the apparent affinity for Na(+) and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K(+)-pNPPase and the apparent affinity for K(+) were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.

Adenosine Triphosphate↗

PubMed2002

A viral RNA that binds ATP and contains a motif similar to an ATP-binding aptamer from SELEX.

The intriguing process of free energy conversion, ubiquitous in all living organisms, is manifested in ATP binding and hydrolysis. ATPase activity has long been recognized to be a capability limited to proteins. However, the presence of an astonishing variety of unknown RNA species in cells and the finding that RNA has catalytic activity have bred the notion that RNA might not be excluded from the group of ATPases. All DNA-packaging motors of double-stranded DNA phages involve two nonstructural components with certain characteristics typical of ATPases. In bacterial virus phi29, one of these two components is an RNA (pRNA). Here we report that this pRNA is able to bind ATP. A comparison between the chemically selected ATP-binding RNA aptamer and the central region of pRNA reveals similarity in sequence and structure. The replacement of the central region of pRNA with the sequence from ATP-binding RNA aptamer produced chimeric aptRNA that is able to both bind ATP and assemble infectious viruses in the presence of ATP. RNA mutation studies revealed that changing only one base essential for ATP binding caused both ATP binding and viral assembly to cease, suggesting that the ATP binding motif is the vital part of the pRNA that forms a hexamer to drive the phi29 DNA-packaging motor. This is the first demonstration of a natural RNA molecule that binds ATP and the first case to report the presence of a SELEX-derived RNA aptamer in living organisms.

Adenosine↗

PubMed2006

ATP-consuming and ATP-generating enzymes secreted by pancreas.

Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid chromatography analysis additionally shows that this enzyme has broad substrate specificity toward other nucleotides, UTP, UDP, ITP, and IDP. In addition, secretion contains ecto-5'-nucleotidase, CD73, further converting [(3)H]AMP to adenosine. Along with highly active hydrolytic enzymes, there were also ATP-generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes release of both ATP-consuming and ATP-generating enzymes into pancreatic juice. This newly discovered richness of secreted enzymes underscores the importance of purine signaling between acini and pancreatic ducts lumen and implies regulation of the purine-converting enzymes release.

5'-Nucleotidase↗

PubMed1975

Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.

Adenosine Diphosphate↗

PubMed1997

Turnover number of Escherichia coli F0F1 ATP synthase for ATP synthesis in membrane vesicles.

The rate of ATP synthesized by the ATP synthase (F0F1-ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Escherichia coli atp wild-type strain. After energization of the membranes with NADH, fractional inactivation of F0F1 by the covalent inhibitor N,N'-dicyclohexylcarbodiimide allowed the rate of ATP synthesis/mol remaining active ATP synthase complexes to increase; the active ATP synthase complexes were calculated using ATP hydrolysis rates as the defining parameter. In addition, variation of the assay temperature revealed an increase of the ATP synthesis rate up to a temperature of 37 degrees C, the optimal growth temperature of E. coli. In parallel, the amount of F0F1 complexes present in membrane vesicles was determined by immunoquantitation to be 3.3 +/- 0.3% of the membrane protein for cells grown in rich medium and 6.6 +/- 0.3% for cells grown in minimal medium with glycerol as sole carbon and energy source. Based on these data, a turnover number for ATP synthesis of 270 +/- 40 s(-1) could be determined in the presence of 5% active F0F1 complexes. Therefore, these studies demonstrate that the ATP synthase complex of E. coli has, with respect to maximum rates, the same capacity as the corresponding enzymes of eukaryotic organells.

Adenosine Triphosphate↗

PubMed1985

Actions of ATP and alpha, beta-methylene ATP on neuromuscular transmission and smooth muscle membrane of the rabbit and guinea-pig mesenteric arteries.

In the rabbit mesenteric artery, adenosine triphosphate (ATP), showed two actions on the membrane potential of muscle cells: low concentrations (1-10 microM) hyperpolarized and high concentrations (greater than or equal to 50 microM) depolarized the membrane. Both changes in the potential were accompanied by increases in ionic conductance. In the rabbit mesenteric artery, alpha, beta-methylene ATP (MeATP), (greater than or equal to 30 nM) depolarized the muscle membrane at a lower concentration than ATP (greater than or equal to 50 microM), and increased the ionic conductance of the membrane. The depolarization induced by ATP was prevented by low concentrations of MeATP, but the hyperpolarization was retained. Furthermore, the hyperpolarization was not affected by theophylline (10 microM). In the guinea-pig mesenteric artery, ATP and MeATP depolarized and increased the ionic conductance of muscle membrane, but to depolarize the membrane, higher concentrations of both agents were required, compared to those in the rabbit mesenteric artery. In the mesenteric arteries from both species, perivascular nerve stimulation evoked excitatory junction potentials (e.j.ps). In both tissues, MeATP inhibited the amplitude of e.j.ps at lower concentrations than did ATP, and both agents had more potent inhibitory actions on rabbit than on guinea-pig. The inhibition of e.j.p. induced by low concentrations of these agents showed no relationship to depolarization, but the inhibition induced by high concentrations was paralleled by depolarization and increase in ionic conductance of the membrane. In the rabbit mesenteric artery, overflow of noradrenaline (NA) and its metabolite (3,4-dihydroxyphenylglycol; DOPEG) produced by perivascular nerve stimulation was examined. ATP (0.1 mM) but not MeATP (0.1 microM) reduced the overflow of NA, whereas both agents had no effect on the overflow of DOPEG. Exogenously applied high concentrations of NA (greater than or equal to 3 microM) depolarized the muscle membrane in both species. These NA-induced depolarizations were not affected by treatment with ATP or MeATP. It is concluded that, in the rabbit mesenteric artery, ATP is more likely to be involved in generation of e.j.ps than is NA. A similar interpretation in the guinea-pig mesenteric artery is complicated by the depolarization produced by high concentrations of ATP or MeATP.

Adenosine Triphosphate↗

PubMed2006

Inhibition of ATP hydrolysis by thermoalkaliphilic F1Fo-ATP synthase is controlled by the C terminus of the epsilon subunit.

The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).

Adenosine Triphosphate↗

PubMed1991

Activation of cardiac ATP-sensitive K+ current during hypoxia: correlation with tissue ATP levels.

Although previous work has implicated activation of ATP-sensitive K+ currents (IK,ATP) in action potential duration (APD) shortening and increased cellular K+ efflux during hypoxia, ischemia, and metabolic inhibition, no prior study has directly assessed the tissue levels of ATP at which IK,ATP activates in intact cardiac muscle. Accordingly, we correlated changes in tissue high-energy phosphate levels during substrate-free hypoxia with activation of IK,ATP in intact voltage-clamped rabbit papillary muscles. During 10 min of hypoxia, the outward K+ current measured in response to a voltage-clamp pulse step from -50 to 0 mV increased from 8.57 +/- 0.27 to 15.67 +/- 1.41 microA (P less than 0.05, n = 6), and APD decreased from 452 +/- 54 to 292 +/- 56 ms (P less than 0.05, n = 6). Glibenclamide (10 microM), a specific IK,ATP blocker, prevented both of these changes. In a parallel set of experiments, papillary muscles were freeze-clamped and assayed for tissue ATP. In these muscles, 10 min of hypoxia resulted in a comparable degree of APD shortening (441 +/- 24 to 297 +/- 18 ms, P less than 0.05, n = 12), and tissue ATP levels fell from 13.2 +/- 1.3 to 9.7 +/- 0.7 mumol/g dry wt (P less than 0.05, n = 12). These results directly demonstrate that IK,ATP is activated and causes APD shortening during hypoxia in intact cardiac muscle despite only a modest (approximately 25%) decline in tissue ATP content.

Action Potentials↗

PubMed1990

Interrelation between pinacidil and intracellular ATP concentrations on activation of the ATP-sensitive K+ current in guinea pig ventricular myocytes.

The patch-clamp technique was used to study the relation between pinacidil and intracellular ATP concentration [( ATP]i) on the activation of the outward K+ current in guinea pig ventricular myocytes. Pinacidil shortened the action potential duration, exhibiting stronger effect at 2 mM [ATP]i than at 5 mM [ATP]i. Pinacidil at 5 microM or higher concentrations activated the time-independent outward current at potentials positive to -80 mV, and the pinacidil-activated current was suppressed by increasing [ATP]i from 2 to 5 mM. The dose-response curve of pinacidil at different [ATP]i showed a shift to the right and a depression of the maximum response at increased [ATP]i. The pinacidil-induced shortening of the action potential duration and outward current were inhibited by application of 0.3-1.0 microM glibenclamide. In single-channel current recordings, pinacidil activated the intracellular ATP-sensitive K+ channel current without changing the unitary amplitude, and increased open probability of the channel, an effect dependent on [ATP]i. The pinacidil-activated single-channel current was blocked by glibenclamide. These results prove the notion that pinacidil activates the ATP-sensitive K+ channel current, which explains the action potential shortening in cardiac cells after application of pinacidil.

Action Potentials↗

PubMed1998

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets.

Insulin secretion by isolated islets of Langerhans from 19 human donors (9 women and 10 men) was studied in vitro to test the hypothesis that human islets contain both the K(ATP) channel-dependent and the K(ATP) channel-independent signaling pathways. The results demonstrated the presence of both of these major pathways of glucose signaling. Thus, insulin secretion was stimulated by high glucose concentrations, by the sulfonylurea tolbutamide, and by a depolarizing concentration of potassium chloride. Diazoxide, which activates the K(ATP) channel, completely blocked the stimulation of release by glucose. Stimulation of insulin release by tolbutamide, which inhibits the K(ATP) channel and depolarizes the beta-cell, and inhibition of glucose-stimulated release by diazoxide, which activates the channel and repolarizes the beta-cell, confirm the involvement of the K(ATP) channel-dependent pathway in glucose signaling. The participation of the K(ATP) channel-independent pathway in the stimulation of insulin release by glucose was demonstrated for the first time in human islets. This was done in two ways. The first method, in the presence of diazoxide, blocked the action of glucose on the K(ATP) channel in combination with a depolarizing concentration of KCl to raise [Ca2+]i. Under these conditions, glucose stimulated insulin release. A second method to demonstrate the involvement of the K(ATP) channel-independent pathway was to close the K(ATP) channels with tolbutamide. Again, with no possibility of further action on the K(ATP) channel, glucose stimulated insulin release. In a final series of experiments, glucose-stimulated insulin release was profoundly inhibited by somatostatin, clonidine, and prostaglandin E2, but not by galanin.

Adenosine Triphosphate↗

PubMed2005

Diabetes and insulin secretion: the ATP-sensitive K+ channel (K ATP) connection.

The ATP-sensitive K+ channel (K ATP channel) senses metabolic changes in the pancreatic beta-cell, thereby coupling metabolism to electrical activity and ultimately to insulin secretion. When K ATP channels open, beta-cells hyperpolarize and insulin secretion is suppressed. The prediction that K ATP channel "overactivity" should cause a diabetic state due to undersecretion of insulin has been dramatically borne out by recent genetic studies implicating "activating" mutations in the Kir6.2 subunit of K ATP channel as causal in human diabetes. This article summarizes the emerging picture of K ATP channel as a major cause of neonatal diabetes and of a polymorphism in K ATP channel (E23K) as a type 2 diabetes risk factor. The degree of K ATP channel "overactivity" correlates with the severity of the diabetic phenotype. At one end of the spectrum, polymorphisms that result in a modest increase in K ATP channel activity represent a risk factor for development of late-onset diabetes. At the other end, severe "activating" mutations underlie syndromic neonatal diabetes, with multiple organ involvement and complete failure of glucose-dependent insulin secretion, reflecting K ATP channel "overactivity" in both pancreatic and extrapancreatic tissues.

Adenosine Triphosphate↗

PubMed2000

Covalent modification of the catalytic sites of the H+-ATPase from chloroplasts and 2-nitreno-ADP. Modification of the catalytic site 1 (tight) and catalytic sites 1 and 2 together impairs both uni-site and multi-site catalysis of ATP synthesis and ATP hydrolysis.

After isolation and purification, the H+-ATPase from chloroplasts, CF0F1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-32P]ADP leads to tight binding of azidonucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of 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-32P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF0F1 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 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF0F1. 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 derivatised CF0F1 and with non-derivatised CF0F1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

Adenosine Diphosphate↗

PubMed2000

Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts with 2-nitreno-ADP. Modification of the catalytic site 1 (tight) and catalytic sites 1 and 2 together impairs both uni-site and multi-site catalysis of ATP synthesis and ATP hydrolysis.

After isolation and purification, the H(+)-ATPase from chloroplasts, CF(0)F(1), contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]ADP leads to tight binding of azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of 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. 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, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. 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 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF(0)F(1). Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF(0)F(1). 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 derivatised CF(0)F(1) and with non-derivatised CF(0)F(1). ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

Journal Article↗

PubMed1987

Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump.

Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases (sodium azide, 20 mM, or oligomycin, 10 micrograms/ml) had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer (pH 5.5), proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect. Disrupted granules only converted proinsulin to a limited extent, and neither ATP nor the inhibitors affected conversion. It is therefore suggested that ATP stimulation of proinsulin conversion in isolated, intact, beta-granules is secondary to intragranular acidification by an ATP-dependent proton pump (reflecting the low pH optimum for proinsulin conversion), rather than ATP dependence of converting activity per se.

Adenosine Triphosphatases↗

PubMed1997

Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

Adenosine↗

PubMed1997

Hepatocellular ATP-binding cassette protein expression enhances ATP release and autocrine regulation of cell volume.

In a model liver cell line, recovery from swelling is mediated by a sensitive autocrine pathway involving conductive release of ATP, P2 receptor stimulation, and opening of membrane Cl- channels (Wang, Y., Roman, R. M., Lidofsky, S. D., and Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release are not known. Based on evidence that certain ATP-binding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ABC proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins. In both cell types, Cl- current activation (ICl-swell) and volume recovery following swelling were dependent on conductive ATP efflux. The rate of volume recovery was approximately 6-fold faster in HTC-R cells compared with HTC cells. This effect is likely due to enhanced ABC protein-dependent ATP release since (i) ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 microM verapamil and 15 microM cyclosporin A); (ii) swelling-induced Cl- current density was similar in both cell types (approximately -50 pA/pF; not significant); and (iii) ATP conductance measured by whole-cell techniques was increased approximately 3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells exhibited enhanced survival during hypotonic stress. By modulating ATP release, hepatic ABC proteins may play a key role in the cellular pathways coupling changes in cell volume to ion permeability and secretion.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1971

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase.

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.

ATP Citrate (pro-S)-Lyase↗

PubMed2001

Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences.

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.

ATP-Binding Cassette Transporters↗