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PubMed1994

The neuronal transport mechanism for noradrenaline is not influenced by the ATP analogue ATP gamma S.

Noradrenaline is taken up in noradrenergic neurones via a desipramine-sensitive transport system (uptake1). There are reports which describe the activity of uptake1 being increased by extracellular ATP and ATP gamma S. This was attributed to a protein kinase-mediated mechanism. We reevaluated this phenomenon since a modulation of uptake1 by extracellular ATP would be of great physiological relevance. In clonal rat pheochromocytoma cells (PC12) we determined the kinetic parameters, Km and Vmax, of noradrenaline transport in the absence and presence of ATP gamma S. The addition of 0.1 mumol/L ATP gamma S had no effect on initial rates of specific noradrenaline transport. Only a slightly increased non-specific noradrenaline flux in the presence of ATP gamma S could be observed. On the basis of our results, there is no evidence for the modulation of the uptake1 carrier by ATP gamma S.

Adenosine Triphosphate↗

PubMed1983

Adenosine 5'-triphosphate (ATP)-mediated stimulation and suppression of DNA synthesis in lymphoid cells. II. Suppressive effect of ATP on murine T-cell functions.

The suppressive effects of ATP on murine T-cell functions were studied. The suppressive effects of ATP as well as adenosine on the DNA synthesis of spleen cells are due to the presence of mature T-cells, because ATP has no suppressive effect on athymic nu/nu spleen cells. Further characterization of the cells which are responsible for ATP-mediated suppression of DNA synthesis revealed that the cells are nylon wool-adherent T-cells and PHA-reactive T-cells. In addition, the suppressive effects of ATP on both spontaneous and mitogen-induced proliferative responses are stronger than that of adenosine, and T-cells are more sensitive to ATP than B-cells. The observation that both ATP and adenosine have unique effects on T-cells compared to B-cells may contribute toward explaining why patients with severe combined immunodeficiency (SCID) associated with adenosine deaminase (ADA) deficiency have greater T-cell than B-cell abnormalities.

Adenosine↗

PubMed2006

Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function.

The Rad51 recombinase polymerizes on ssDNA to yield a right-handed nucleoprotein filament, called the presynaptic filament, that can search for homology in duplex DNA and pair the recombining DNA molecules to form a DNA joint. ATP is needed for presynaptic filament assembly and homologous DNA pairing, but the roles of ATP binding and ATP hydrolysis in the overall reaction scheme have not yet been clearly defined. To address this issue, we have constructed two mutants of hRad51, hRad51 K133A and hRad51 K133R, expressed these mutant variants in Escherichia coli, and purified them to near homogeneity. Both hRad51 mutant variants are greatly attenuated for ATPase activity, but hRad51 K133R retains the ability to protect DNA from restriction enzyme digest and induce topological changes in duplex DNA in an ATP-dependent manner, whereas the hRad51 K133A variant is inactive. With biochemical means, we show that the presynaptic filament becomes greatly stabilized when ATP hydrolysis is prevented, leading to an enhanced ability of the presynaptic filament to catalyze homologous pairing. These results help form the basis for understanding the functions of ATP binding and ATP hydrolysis in hRad51-mediated recombination reactions.

Adenosine Triphosphatases↗

PubMed2006

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome.

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.

Adenosine Triphosphate↗

PubMed2005

K(ATP) channels and preconditioning: a re-examination of the role of mitochondrial K(ATP) channels and an overview of alternative mechanisms.

Preconditioning by one or several brief periods of ischemia activates an endogenous cardioprotective program that increases the resistance of cardiomyocytes to injury by subsequent prolonged periods of ischemia. Ischemic preconditioning can be mimicked by K(+) channel openers and various other substances, a phenomenon termed pharmacological preconditioning. Initially, ischemic preconditioning has been ascribed to the opening of ATP-sensitive K(+) channels at the surface membrane of cardiomyocytes. Since 1997, numerous publications have implicated mitochondrial ATP-sensitive K(+) channels (mK(ATP)) as a major trigger and/or end effector of preconditioning. Diazoxide has been suggested to be a specific activator of mK(ATP) channels, and the substituted fatty acid 5-hydroxydecanoate (5-HD) has been suggested to be a specific inhibitor. However, diazoxide and 5-HD have multiple K(+)-channel-independent actions, and the experimental evidence for an obligatory role of mK(ATP) channels in preconditioning, or even their existence, remains inconclusive. In contrast, surface K(ATP) channels have been well characterized, and we summarize the evidence suggesting that they make a major contribution to preconditioning. We also discuss a number of other factors involved in preconditioning: (1) generation of reactive oxygen species, (2) impairment of fatty acid metabolism, and (3) opening of the mitochondrial permeability transition pore. In the light of these emerging concepts, we critically re-examine the evidence for and against a role of mK(ATP) channels in ischemic and pharmacological preconditioning.

Animals↗

PubMed2001

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F(0)F(1)-ATP synthase is absent from about half of chromatophores.

F(0)F(1)-ATP synthase (H(+)-ATP synthase, F(0)F(1)) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P(i)). Structurally the enzyme consists of a membrane-embedded proton-translocating F(0) portion and a protruding hydrophilic F(1) part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F(0)F(1) (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F(1) (efrapeptin) and of F(0) (venturicidin), we decomposed the kinetics of the total proton flow through F(0)F(1) into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F(0). Taking the coupled proton flow, we calculated the H(+)/ATP ratio; it was found to be 3.3+/-0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1+/-0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F(0)F(1) dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P(i) led to an increase in the fraction of chromatophores with at least one de-coupled F(0)(F(1)). The disclosed fraction of chromatophores that lacked proton-conducting F(0)(F(1)) (approx. 40% of the total amount) remained constant upon these treatments.

Adenosine Diphosphate↗

PubMed1991

Hexokinase bound to rat brain mitochondria uses externally added ATP more efficiently than internally generated ATP.

The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from rat brain. Initial rates obtained either with ATP generated from oxidative phosphorylation or with ATP added externally were compared. Our results show that the external ATP supports a 2-3-fold higher hexokinase activity than does ATP generated by oxidative phosphorylation under Stage 3 conditions. ATP formed by mitochondrial creatine kinase in the presence of creatine phosphate also supports higher initial rates of glucose phosphorylation than does oxidative phosphorylation. The data suggest that concentrations of ATP present in the cytosol of normal tissue will probably maintain higher rates of glucose phosphorylation than ATP being exported directly from the mitochondrial matrix at maximal State 3 rates.

Adenosine Triphosphate↗

PubMed1997

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation.

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.

Adenosine Diphosphate↗

PubMed2001

Mitochondria are the main ATP source for a cytosolic pool controlling the activity of ATP-sensitive K(+) channels in mouse cardiac myocytes.

OBJECTIVE: The aim was to identify the major ATP source controlling the activity of sarcolemmal K(ATP) channels in ventricular cardiomyocytes. METHODS: K(ATP)-channel current (I(KATP)) was measured with the patch-clamp technique in either the whole-cell (glycogenolysis blocked by 10 mmol/l EGTA), cell-attached, or inside-out configuration. RESULTS: In the absence of any substrate, I(KATP) (amplitude 31+/-4 nA; n=5) appeared spontaneously 520+/-160 s (n=6) after whole-cell access. This latency was shortened by exposure to anoxia (117+/-33 s, n=32) and even more by uncoupling (1-10 micromol/l FCCP; 25+/-3 s; n=13) while the amplitude was unchanged. During metabolic inhibition the latency was remarkably prolonged when the F1F0-ATPase was blocked by oligomycin, suggesting that under those conditions the F1F0-ATPase is the major ATP consumer. Glucose (5.5-20.0 mmol/l) in the bath solution did not affect the amplitude of I(KATP) but prolonged its latency compared to respective substrate-free conditions. However, I(KATP) was blocked immediately by mitochondrial substrates. FCCP also induced large I(KATP) in cell-attached measurements in either the absence or presence of glucose and oligomycin. CONCLUSIONS: The activity of K(ATP) channels in cardiomyocytes of mice is controlled by a cytosolic [ATP] pool for which oxidative phosphorylation is the predominant ATP source.

Adenosine Triphosphate↗

PubMed1998

ATP synthesis by the F1Fo ATP synthase of Escherichia coli is obligatorily dependent on the electric potential.

The H+-translocating F1Fo ATP synthase of Escherichia coli was purified and reconstituted into proteoliposomes. This system catalyzed ATP synthesis when energized by an acid/base transition (pHin = 5.0; pHout = 8.3) with succinate, malonate or maleinate but not with MES as the acidic buffer. Under these experimental conditions an electric potential of 125-130 mV is generated by the diffusion of succinate, probably the monoanionic species, whereas with MES buffer the measured potential was at background level (approximately 5 mV). ATP was also synthesized at pH 7.2 in the absence of a delta pH by applying a K+/valinomycin diffusion potential. The rate of ATP synthesis increased with the potential in an exponential manner with an inflection point at about 70 mV. We conclude from these results that delta pH and delta psi are kinetically unequivalent driving forces for ATP synthesis by the E. coli ATP synthase and that delta psi is a mandatory force for this synthesis. The significance of these findings for the mechanism of ATP synthesis in general is discussed.

Adenosine Triphosphate↗

PubMed2003

Complete DNA sequence of the atp operon of the sodium-dependent F1Fo ATP synthase from Ilyobacter tartaricus and identification of the encoded subunits.

The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F(1)F(o) ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6,992 base pairs with a GC content of 38.1%. The F(1)F(o) ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4,810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na(+)-translocating F(1)F(o) ATP synthases, especially in the F(o) subunits a and c which are directly involved in ion translocation.

Adenosine Triphosphate↗

PubMed1985

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

Actins↗

PubMed2005

Oxygen blocks the reaction of the FixL-FixJ complex with ATP but does not influence binding of FixJ or ATP to FixL.

The RmFixL-RmFixJ oxygen signal transduction system ensures that a cascade of the Sinorhizobium meliloti nitrogen fixation genes is induced as the concentration of O2 drops below 50 microM in symbiotic nodules. Deoxy-RmFixL is a histidine protein kinase that catalyzes a phosphoryl transfer from ATP to the aspartate 54 residue of RmFixJ; RmFixJ is a response regulator that becomes activated as a transcription factor by phosphorylation. Association of O2 with a heme-binding domain in RmFixL triggers a conformational change that inhibits its kinase activity. Here we consider whether this inhibition is achieved by disrupting binding of either of the substrates, i.e., RmFixJ or ATP, to the RmFixL kinase. The ATP affinities of the oxy and deoxy states were compared via competition of ATP against TNP-nucleotide fluorophores. The influence of O2 on formation of the RmFixL-RmFixJ complex was investigated by fluorescence polarization. Oxygen dramatically inhibited the reaction of the RmFixL-RmFixJ complex with ATP but affected neither ATP binding (Kd approximately 100 microM) nor RmFixL-RmFixJ complex formation (Kd approximately 4 microM), indicating that inhibition of the kinase by the oxy-heme in RmFixL is achieved by inactivating the catalytic site, rather than by blocking the association of this enzyme with either of its substrates. An 8-fold enhancement of the rate of reaction of RmFixL with ATP in a deoxy-RmFixL-D54N RmFixJ complex, compared to that in isolated deoxy-RmFixL, exposes the strength of the allosteric effect of RmFixJ on the reaction. These results clarify the mechanistic roles of the signal and regulatory partner in this signal transduction system.

Adenosine Triphosphate↗

PubMed2006

Structure of ATP-bound human ATP:cobalamin adenosyltransferase.

Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B12) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 A crystal structure of ATP bound to hATR refined to an Rfree value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

Adenosine Triphosphate↗

PubMed1985

Evidence that the Mg-dependent low-affinity binding site for ATP and Pi demonstrated on the isolated beta subunit of the F0.F1 ATP synthase is a catalytic site.

Binding sites for one Pi and two ATP or ADP molecules have been identified on the isolated, reconstitutively active beta subunit from the Rhodospirillum rubrum F0.F1 ATP synthase. Chemical modification of this beta subunit by the histidine reagent diethyl pyrocarbonate or by the carboxyl group reagent Woodword's reagent K results in complete inhibition of Pi binding to beta. The same reagents inhibit the binding of ATP to a Mg-dependent low-affinity site but not to a Mg-independent high-affinity site on this beta subunit. The binding stoichiometry of ADP to either site is not affected by these modifications. The beta subunit modified by either one of these reagents retains its capacity to rebind to beta-less chromatophores but not its ability to restore their photo-phosphorylation. These results indicate that the low-affinity Pi binding site on beta is located at the binding site of the gamma-phosphate group of ATP in the Mg-dependent low-affinity nucleotide binding site. This site contains histidine and carboxyl group residues, both of which are required for the binding of Pi and of the gamma-phosphate group of ATP. The same residues must also be involved in the capacity of the isolated beta subunit to restore the catalytic activity of the beta-less ATP synthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated beta subunit of the R. rubrum F0.F1 ATP synthase is the catalytic site of this enzyme complex.

Adenosine Diphosphate↗

PubMed1989

Photolysis of a photolabile precursor of ATP (caged ATP) induces microsecond rotational motions of myosin heads bound to actin.

To test the proposal that ATPase activity is coupled to the rotation of muscle cross-bridges (myosin heads attached to actin), we have used saturation-transfer EPR to detect the rotational motion of spin-labeled myosin heads (subfragment 1; S1) bound to actin following the photolysis of caged ATP (a photoactivatable analog of ATP). In order to ensure that most of the heads were bound to actin in the presence of ATP, solutions contained high (200 microns) actin concentrations and were of low (36 mM) ionic strength. Sedimentation measurements indicated that 52 +/- 2% of the spin-labeled heads were attached in the steady state of ATP hydrolysis during EPR measurements. Five millimolar caged ATP was added to the actin-S1 solution in an EPR cell in the dark, with no effect on the intense saturation-transfer EPR signal, implying a rigid actin-S1 complex. A laser pulse produced 1 mM ATP, which decreased the signal rapidly to a brief steady-state level that indicated only slightly less rotational mobility than that of free heads. After correcting for the fraction of free heads, we conclude that the bound heads have an effective rotational correlation time of 1.0 +/- 0.3 microseconds, which is about 100 times shorter (faster) than that in the absence of ATP. To our knowledge, this is the first direct evidence that myosin heads undergo rotational motion when bound to actin during the ATPase cycle. It is likely that similar cross-bridge rotations occur during muscle contraction.

Actins↗

PubMed1999

Role of walker motif A of RuvB protein in promoting branch migration of holliday junctions. Walker motif a mutations affect Atp binding, Atp hydrolyzing, and DNA binding activities of Ruvb.

Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69). K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of the ruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro. In the presence of Mg(2+), K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg(2+) and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA. All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.

Adenosine Triphosphate↗

PubMed2003

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP.

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.

Animals↗