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PubMed2002

A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins.

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archae. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon from Thermococcus kodakaraensis KOD1 (Lon(Tk)) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA(+) superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that Lon(Tk) was actually a membrane-bound protein. The recombinant Lon(Tk) possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70 degrees C, respectively. Unlike the enzyme from Escherichia coli, we found that Lon(Tk) showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that Lon(Tk) required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, Lon(Tk) degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in Lon(Tk) different from that of its bacterial counterpart.

ATP-Dependent Proteases↗

PubMed2004

How does protein architecture facilitate the transduction of ATP chemical-bond energy into mechanical work? The cases of nitrogenase and ATP binding-cassette proteins.

Transduction of adenosine triphosphate (ATP) chemical-bond energy into work to drive large-scale conformational changes is common in proteins. Two specific examples of ATP-utilizing proteins are the nitrogenase iron protein and the ATP binding-cassette transporter protein, BtuCD. Nitrogenase catalyzes biological nitrogen fixation whereas BtuCD transports vitamin B(12) across membranes. Both proteins drive their reactions with ATP. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated in these proteins, a coarse-grained elastic network model is employed. The analysis shows that subunits of the proteins move against each other in a concerted manner. The lowest-frequency modes of the nitrogenase iron protein and of the ATP binding-cassette transporter BtuCD protein are found to link the functionally critical domains, and these modes are suggested to be responsible for (at least the initial stages) large-scale ATP-coupled conformational changes.

ATP-Binding Cassette Transporters↗

PubMed1981

ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase phosphorylate different sites on ATP-citrate lyase.

ATP-citrate lyase was phosphorylated by highly purified cyclic AMP-independent protein kinase (ATP-citrate lyase kinase) or the catalytic subunit of cyclic AMP-dependent protein kinase. Each kinase phosphorylated ATP-citrate lyase equally but the combination of both kinases increased ATP-citrate lyase phosphorylation additively. When ATP-citrate lyase was phosphorylated with each kinase and partially digested with either L-1-tosylamido-2-phenylmethyl chloromethyl ketone-treated trypsin or Staphylococcus aureus protease followed by electrophoresis of the proteolytic products on sodium dodecyl sulfate-polyacrylamide gels or when the phosphorylated lyase was completely digested by these proteases followed by chromatography and electrophoresis, the results showed that the site phosphorylated by ATP-citrate lyase kinase was different from that phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Only phosphoserine was found in lyase phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase whereas phosphoserine and phosphothreonine were found in ATP-citrate lyase phosphorylated by lyase kinase.

ATP Citrate (pro-S)-Lyase↗

PubMed1983

The synthesis of ATP by the membrane-bound ATP synthase complex from medium 32Pi under completely uncoupled conditions.

Previously, we demonstrated that isolated coupling factor 1 can reversibly synthesize bound ATP from "tightly bound" ADP and medium Pi (Feldman, R I., and Sigman, D. S. (1982) J. Biol. Chem. 25, 1676-1683). In order to ensure that the thermodynamic constants derived are relevant to coupled ATP synthesis, we have also studied the reaction on thylakoid membranes. The ATP synthase complex, uncoupled with 20 mM NH4Cl or 0.3% Triton X-100, synthesizes enzyme-bound ATP in a similar manner to coupling factor 1. The pH optimum is 6, the concentration of medium Pi for 50% saturation is 38 mM, and the equilibrium constant for the formation of ATP from bound ADP and Pi is 0.5. It is concluded that the active site responsible for the reaction is not appreciably altered by the dissociation of coupling factor 1 from the membrane or Fo. Thus, either enzyme form can be used to derive data relevant to the mechanism of ATP synthesis. The ability to measure bound ATP synthesis in an energizable system will allow us to probe the effect of membrane energization on the accumulated bound product.

ATP Synthetase Complexes↗

PubMed2002

Relief of inflammatory pain in rats by local use of the selective P2X7 ATP receptor inhibitor, oxidized ATP.

OBJECTIVE: Oxidized ATP (oATP) is a selective inhibitor of the P2Z/P2X7 ATP receptor for extracellular ATP, which contributes to the antinociceptive effect. This study sought to determine the mechanism by which local administration of oATP is able to relieve inflammatory pain in arthritic rat paws. METHODS: Arthritis was induced in Wistar rats by injections of Freund's complete adjuvant into one hind paw. Nociceptive thresholds were measured before and after local injection of oATP into the inflamed paws. The influence on pain transmission due to the presence of recruited inflammatory cells at the site of inflammation was determined by inhibiting the initial phase of their migration (by intravenous treatment with fucoidin, which blocks the adhesion molecules of the selectin family). ATP intraplantar content was determined in the different experimental conditions. Histologic features of the hind paws were evaluated by using the anti-P2X7 receptor polyclonal antibody. RESULTS: Intraplantar administration of oATP into inflamed paws significantly relieved inflammatory pain. The antinociceptive effect of oATP was independent of the immune-cell recruitment. ATP levels in inflamed tissues were significantly reduced by oATP treatment. A variable presence of P2X7 receptors on cutaneous sensory nerves with respect to the different treatments was observed. Following oATP treatment, there was a reduction in P2X7 expression in the endings of peripheral nerves, as well as in endothelial cells. CONCLUSION: Oxidized ATP inhibits inflammatory pain in arthritic rats by inhibition of the P2X7 receptor for ATP, which is localized on nerve terminals.

Adenosine Triphosphate↗

PubMed1996

The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis.

During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.

Adenosine Triphosphate↗

PubMed2000

Kinetic modeling of ATP synthesis by ATP synthase and its mechanistic implications.

Based on the torsional mechanism of ATP synthesis by ATP synthase, a kinetic scheme has been developed in this work. The scheme considers adenine nucleotide transport, binding of substrates ADP and P(i), unbinding of product ATP, and ATP synthesis. This kinetic scheme has been analyzed mathematically, and a kinetic model has been obtained to explain the experimentally observed hyperbolic Michaellian dependence of the rate of ATP synthesis on the ADP concentration by ATP synthase under physiological steady-state operating conditions. The principal results of the kinetic model have been compared with the experimental data and an estimate of the enzymological kinetic parameters V(max), K(M), and K(I) has been determined. Mechanistic implications arising from further analysis of the kinetic model have been discussed. These biological implications provide deep insight into the sequence of events leading to ATP synthesis.

Adenosine Diphosphate↗

PubMed1989

The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells.

The ATP- and sulphonylurea-sensitivity of the ATP-sensitive K-channel was measured in human pancreatic B cells. In inside-out patches, half-maximal inhibition of channel activity was produced by 10 mumol/l ATP (with 2 mM Mg2+) and ATP-inhibition was partially antagonised by ADP. A significantly lower sensitivity to ATP was found in whole-cell recordings. Tolbutamide inhibited whole-cell ATP-sensitive K-currents half-maximally at 18 mumol/l; the sensitivity to tolbutamide was somewhat less in the inside-out patch. Ca-activated K-channels were unaffected by tolbutamide (10 mmol/l). These results resemble those found for rodent B cells and suggest that sulphonylureas exert their therapeutic effects in Type 2 (non-insulin dependent) diabetes by inhibition of the ATP-sensitive K-channel.

Adenosine Triphosphate↗

PubMedDate not supplied

Ischaemic ATP degradation studied by HPLC and 31P-NMR spectroscopy: do the two techniques observe the same ATP pools?

31P-NMR spectroscopy has become the major tool for studying myocardial high energy phosphates. Conflicting results concerning NMR visibility of ATP in ischaemic myocardium were reported. A detailed study was undertaken to resolve this controversy. After cardioplegic arrest, canine hearts were excised and preserved for 24 h at 1 degree C (group 1) or for 6h at 23 degrees C (group 2). ATP breakdown was followed by 31P-NMR spectroscopy in a transmural piece of the anterior wall introduced in the NMR magnet, and by HPLC analysis using serial transmural biopsies from the rest of the anterior wall. At both temperatures, identical relative ATP decay curves were obtained, whether measured by NMR or by HPLC. Absolute quantification of ATP was carried out after varying periods of ischaemia at 1 degree C. The NMR-measured ATP concentration was 106 +/- 8% of the ATP concentration determined by HPLC. From our experiments, we conclude that ATP visibility for 31P-NMR spectroscopy is complete and constant during prolonged periods of hypothermic ischaemia in canine hearts.

Adenosine Triphosphate↗

PubMed1999

Kinetics of PCr to ATP and beta-ATP to beta-ADP phosphoryl conversion are modified in working rat skeletal muscle after training.

Kinetics of phosphoryl transfers from PCr to gamma-ATP and from beta-ATP to beta-ADP were measured by magnetization transfer in an in vivo 31P NMR experiment in working rat skeletal hind leg muscles. Two groups were examined. One group was submitted to a 6-week training program of treadmill running. The other group was composed of sedentary animals. Metabolic oxidative capacity and mechanical performance were improved greatly by training as shown previously. Phosphoryl transfer of PCr-->gamma-ATP or beta-ATP-->beta-ADP total fluxes were identical in resting trained and untrained muscles. Under stimulation, the flux of creatine kinase transfer was significantly inhibited by 23% compared with resting level in untrained muscles; by contrast, it was not inhibited and maintained at the high resting level in trained muscles. Thus physiological changes probably linked to a decrease of the production of anions, which could inhibit creatine kinase, were able to maintain creatine kinase flux. The flux of beta-ATP to beta-ADP transfer were enhanced largely in working muscles from 1.4+/-0.8 and 2+/-0.8 at rest to 4+/-1.6 and 6.6+/-2.7 mM s(-1) for untrained and trained muscles respectively; the effect was more pronounced in trained than in untrained muscles. These results showed an acceleration of phosphoryl turnover in working muscles after training, which could contribute to improve oxidative and mechanical performances. Such kinetic measurements of phosphoryl conversion may provide information on ATP turnover in pathophysiologic situations where ADP accumulates because of impaired ATP synthesis (mitochondrial myopathies, lower perfusion level).

Adenosine Diphosphate↗

PubMed1983

ATPase of bovine heart mitochondria. Modulation of ITPase activity by ATP, ADP, acetyl ATP and acetyl AMP.

(1) Mitochondrial ATPase (F1) is influenced by specific nucleotides in its kinetic behavior towards its substrates. In this work, initial hydrolysis rates, as well as continuous reaction progress, were measured by recording proton production (equivalent to triphosphate hydrolysis). (2) After preincubation with ATP, F1 hydrolyzes MgITP partly as if it were MgATP, with respect to temperature dependence and 2,4-dinitrophenol inhibition/stimulation. (3) Acetyl ATP is a competitive inhibitor versus ATP on the F1-ATPase. With F1 which has been freed of ambient ATP by repeated precipitations with ammonium sulfate the Ki of acetyl ATP is 400 nM. (4) F1-ATPase which was depleted of bound nucleotides in the presence of glycerol (Garret, N.E. and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647) was preincubated with ADP and acetyl ATP. These preparations were assayed for hydrolytic activity with MgITP as substrate. Compared to a nonpreincubated control enzyme, the hydrolysis with these preparations was first stimulated, then inhibited. This stimulation/inhibition effect is most pronounced at 10 degrees C, but is also observed at 20 degrees C. (5) When nucleotide-depleted enzyme is preincubated with acetyl AMP, its ability to hydrolyze MgITP slowly decreases to approx. 50% after 60 min. This effect is reversed by further preincubation with acetyl ATP. It is speculated that under appropriate conditions AMP may exist or arise in a buried position on F1-ATPase, and act there as an inhibitor of MgITP hydrolysis.

Adenine Nucleotides↗

PubMed1988

Current transients generated by the Na+/K+-ATPase after an ATP concentration jump: dependence on sodium and ATP concentration.

Planar membrane fragments containing a high density of oriented Na+/K+-ATPase molecules are bound to planar lipid bilayers. ATP is released in the aqueous solution within milliseconds from an inactive, photolabile precursor ('caged ATP') by an intense light flash. By this ATP-concentration jump a large number of pump molecules is activated almost simultaneously. Charge translocation in the pump molecule results in a voltage transient which is recorded in the external measuring circuit. From the voltage signal, the intrinsic pump current Ip(t) can be evaluated using information on the circuit parameters of the compound membrane system. The pump current Ip(t) is compared with the results of numerical simulations of a reaction cycle derived from the Post-Albers reaction scheme combined with the photochemical release reaction of caged ATP. The time course of Ip can be satisfactorily fitted using kinetic parameters of the Na+/K+-ATPase from the literature. The dependence of Ip on sodium concentration cNa can be described using a single set of kinetic parameters in which only cNa is varied. Ip as a function of cNa is well fitted by a first-order Michaelis-Menten type equation with Km approximately equal to 4 mM. This finding is consistent with the assumption that two sodium binding sites have a high affinity and that a third site of lower affinity is rate limiting. The ATP concentration dependence of Ip is studied by varying the concentration of caged ATP in the solution and the yield of photochemical release of ATP.

Adenosine Triphosphate↗

PubMed1993

Signal-transduction mechanisms of ATP-stimulated phosphatidylcholine secretion in rat type II pneumocytes: interactions between ATP and other surfactant secretagogues.

ATP stimulates phosphatidylcholine secretion in type II cells, an effect that is mediated by both adenosine A2 receptors coupled to adenylate cyclase and P2 receptors coupled to phosphoinositide-specific phospholipase C. Activation of these effector enzymes leads to formation of cAMP, diacylglycerols and inositol trisphosphate (IP3). cAMP in turn activates cAMP-dependent protein kinase, diacylglycerols activate protein kinase C and IP3 promotes Ca2+ mobilization. To further investigate the signal-transduction mechanisms mediating the ATP effect, we examined its action in combination with that of other surfactant secretagogues: 5'(N-ethylcarboxyamido)adenosine (NECA), a A2 agonist that activates adenylate cyclase; TPA (12-O-tetradecanoylphorbol-13-acetate), a direct activator of protein kinase C; and ionomycin, an ionophore that increases intracellular Ca2+. The effects of NECA, TPA and ionomycin were additive and thus consistent with independent signaling mechanisms. However, the effects of all combinations of three or four secretagogues that contained ATP were 10-20% less than additive. This suggested that ATP and other secretagogues act via common mechanisms. Calmodulin antagonists decreased the effects of ionomycin and ATP by approx. 60% and 30%, respectively, but did not decrease the effects of NECA, terbutaline or TPA. Complete inhibition of the effect of ATP was achieved with a combination of a calmodulin antagonist, an A2 antagonist and a protein kinase C inhibitor. These and previous data suggest that the stimulatory effect of ATP on phosphatidylcholine secretion in type II cells is mediated by three signal-transduction mechanisms: activation of cAMP-dependent protein kinase; activation of protein kinase C; and a calmodulin-dependent mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G.

Rhodamine 6G (3 microM) effectively inhibited delta pH-driven ATP synthesis in Methanobacterium thermoautotrophicum while delta pNa-driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg(2+)-stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis-driven ATP synthesis at pH 6.8 of cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis-driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium-motive force-driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively in alkaline conditions and it might be the sole ATP synthesizing system when the proton-motive force-supported ATP synthesis is inhibited by rhodamine 6G.

Adenosine Triphosphatases↗

PubMed1994

Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G.

Rhodamine 6G (3 microM) effectively inhibited delta pH-driven ATP synthesis in Methanobacterium thermoautotrophicum while delta pNA-driven ATP synthesis was not affected by it. Rhodamine 6G inhibited Mg(2+)-stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor. Methanogenesis-driven ATP synthesis at pH 6.8 of the cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl. On the other hand, the methanogenesis-driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl. The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl. These results indicate that sodium-motive force-driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively at alkaline conditions and it might be the sole ATP synthesizing system when the proton motive force-supported ATP synthesis is inhibited by rhodamine 6G.

Adenosine Triphosphatases↗

PubMed1994

Mitochondrial ATP hydrolysis and ATP depletion in thymocytes and Ehrlich ascites carcinoma cells.

When Ehrlich ascites carcinoma (EAC) cells and thymocytes were treated with uncoupler or rotenone in glucose-free medium, rapid ATP depletion was observed in both types of the cells. Oligomycin slowed down ATP loss in thymocytes, but not in EAC cells. Thus, mitochondrial ATP hydrolysis appears to be significant in deenergized thymocytes in contrast to EAC cells, in which other ATP consuming reactions were prevailing. Complete deenergization of mitochondria by uncoupler or rotenone in these cells resulted in inactivation of mitochondrial ATPase by 65-75%. The effect was observed after complete and rapid (20-30 s) disruption of the cells with detergent, Lubrol WX. ATPase was blocked by the specific inhibitor protein (IF1) as it was shown by the studies on reactivation of this enzyme. When respiration is blocked but ATP content is supported by glycolysis, mitochondrial ATPase is not suppressed by IF1, and maintains the energization of mitochondria. It is concluded that under complete de-energization of mitochondria IF1 significantly inhibits mitochondrial ATP hydrolysis and may slow down ATP loss in thymocytes and EAC cells.

Adenosine Triphosphatases↗

PubMed1995

ATP synthesis catalyzed by the mitochondrial F1-F0 ATP synthase is not a reversal of its ATPase activity.

The ADP(Mg2+)-deactivated oligomycin-sensitive F1-F0 ATPase of coupled submitochondrial particles treated with the substoichiometric amount of oligomycin was studied to test whether ATP synthesis and hydrolysis proceed in either direction through the same intermediates. The initial rates of ATP hydrolysis, oxidative phosphorylation, ATP-dependent, succinate-supported NAD+ reduction, and ATP-induced delta microH+ generation were measured using deactivated ATPase trapped by azide [Biochem. J. (1982) 202, 15-23]. Three ATP consuming reactions were strongly inhibited when azide was present in the assay mixtures, whereas ATP synthesis was not altered by azide. The unidirectional effect of azide is not consistent with three alternating binding sites mechanism operating in ATP synthesis and support our hypothesis on the existence of nucleotide(Mg2+)-controlled 'synthase' and 'hydrolase' states of the mitochondrial F1-F0 ATPase.

Adenosine Triphosphate↗

PubMed1992

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.

Adenosine Triphosphate↗