Value Of ATP Research Articles

Effects of anions on ATP-activated nonselective cation current in NG108-15 cells.

Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS-) than in Cl- external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density (Imax) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent Imax decreased in the order of aspartic acid (Asp-) > MS- > F- > Cl- > Br- > or = I-. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp- < F- < MS- < Br- < Cl- < I-. Thus both Imax and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl- was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.

Growth energetics and metabolic fluxes in continuous cultures of Penicillium chrysogenum

Continuous cultures of the penicillin producing fungus Penicillium chrysogenum have been analyzed with respect to the macromolecular composition of the mycelium. All cultivations were carried out using a chemically defined medium with glucose as the growth limiting component. Biomass was harvested at steady state and analyzed for proteins, lipids, RNA, DNA, and carbohydrates. Carbohydrates present in the cell wall i.e., glucans and chitin, and carbohydrates serving as storage materials i.e., glycogen, were measured. It was observed that the levels of DNA and lipids are relative constant, whereas the proteins and stable RNA levels increase with the specific growth rate and the total amount of carbohydrates decreases with the specific growth rate. Glycogen is only present in small amounts, decreasing with the specific growth rate. As an average the measured macromolecules account for 77 ± 2% (w/w) of the biomass. On the basis of estimations of the metabolic costs for biosynthesis and polymerization of the different macromolecules the total ATP and NADPH requirements for cell biosynthesis from glucose and inorganic salts i.e., Y xATP,growth and Y xNADPH' have been quantified. The biosynthesis of l g biomass was calculated to require 39.9 mmol of ATP and 7.5 mmol of NADPH when cytosolic acetyl-CoA is formed from citrate by citrate lyase and oxaloacetate is recycled back into the TCA cycle. Other pathways of acetyl-CoA biosynthesis have been considered. The calculations show that the different biosynthetic routes for generating cytosolic acetyl-CoA have a significant influence on the theoretical value of ATP and NADPH requirements for cell biosynthesis. Combining a detailed stoichiometric model for growth and product formation of P. chrysogenum with experimental data on the macromolecular composition of P. chrysogenum and precise measurements of substrate uptake and product formation the intracellular flux distribution was calculated for different cultivation conditions.

Comparative energetics of glucose and xylose metabolism in ethanologenic recombinant Escherichia coli B.

This study compared the anaerobic catabolism of glucose and xylose by a patented, recombinant ethanologenic Escherichia coli B 11303:pLOI297 in terms of overall yields of cell mass (growth), energy (ATP), and end product (ethanol). Batch cultivations were conducted with pH-controlled stirred-tank bioreactors using both a nutritionally rich, complex medium (Luria broth) and a defined salts minimal medium and growth-limiting concentrations of glucose or xylose. The value of gamma ATP was determined to be 9.28 and 8.19 g dry wt cells/mol ATP in complex and minimal media, respectively. Assuming that the nongrowth-associated energy demand is similar for glucose and xylose, the mass-based growth yield (Yx/s, g dry wt cells/g sugar) should be proportional to the net energy yield from sugar metabolism. The value of Yx/s was reduced, on average, by about 50% (from 0.096 g/g glu to 0.51 g/g xyl) when xylose replaced glucose as the growth-limiting carbon and energy source. It was concluded that this observation is consistent with the theoretical difference in net energy (ATP) yield associated with anaerobic catabolism of glucose and xylose when differences in the mechanisms of energy-coupled transport of each sugar are taken into account. In a defined salts medium, the net ATP yield was determined to be 2.0 and 0.92 for glucose and xylose, respectively.

Binding of the radioligand [ 3Sadenosine 5'- O-(2-thiodiphosphate) and intracellular calcium response in rat liver parenchymal cells

The use of the radioligand [ 35S]adenosine 5'- O-(2-thiodiphosphate) (ADP β 35S) for the determination of P 2y-purinoceptors on turkey erythrocyte membranes has recently been described. In the present study, we were able to demonstrate specific binding of this radioligand in intact rat liver parenchymal cells. Within 10 min a thermodynamic equilibrium was obtained which lasted for 25 min with a subsequent decline. Displacement studies with several nucleotides were performed yielding K i values of 1.5 ± 0.47 μM for UTP, 1.8 ± 0.35 μM for adenosine 5'- O-(2-thiodiphosphate) (ADPβS), 31 ± 6.2 μM for ATP and 35 ± 6.1 μM for GTP. In addition, we showed that ADP β 35S is highly resistant to degradation by ecto-nucleotidases, with only 14.5 ± 1.4% of total ADP β 35S present being degraded after 1 hr, and that the binding of ADP β 35S to its binding sites was modulated by EDTA. The K i value of ATP shifted to 8.1 ± 1.2 μM upon the addition of 1 mM EDTA to the incubation medium. In these rat liver parenchymal cells all nucleotides promoted calcium entry in a dose-dependent manner with ec 50 values of 3.5 ± 0.22 μM for UTP, 20.7 ± 3.1 μM for ATP, 38.3 ± 6.4 jμM for ADPβS and 73.6 ± 13.7 μM for GTP, with GTP being a partial agonist. Based on the data derived from the present study we discuss the possible correlation between binding and functional experiments and conclude that the described receptor resembles most closely the P 2u-purinoceptor and/or “nucleotide receptor”, in that UTP is at least as active as ATP.

Ca 2+/calmodulin-dependent protein kinase II from hen brain : Purification and characterization

Ca 2+/calmodulin-dependent protein kinase II (CaM-kinase II) has been purified from hen whole brain. The enzyme was purified 3000-fold using phosphocellulose and calmodulin-Agarose column chromatography. The specific activity was 200 nmol/min/mg protein. Microtubule associated protein-2 (MAP-2) was used as a substrate to assess the activity of the enzyme during purification and for its characterization. CaM-kinase II consisted of α and β/β′ subunits of molecular weights 46,000 and 55,000/ 52,000, respectively. The ratio of α to β/β′ subunits was 3:1 in the enzyme purified from the whole brain. The enzyme exhibited broad substrate specificity and phosphorylated myelin basic protein, MAP-2, histone II, histone VIII, casein, tubulin, myosin light chains, glycogen synthase, and phosvitin in decreasing order. Phosphorylase b was phosphorylated at a negligible rate. Autophosphorylation of CaM-kinase II for 10 min in the presence of calcium and calmodulin decreased its total activity to 33%, and calcium/calmodulin-independent activity reached 30% after 1 min and then dropped to 14% after 10 min of autophosphorylation. The K m value of ATP was 19 ± 1.3 μM, and the K 0.5 values of calcium and calmodulin were 4.4±0.5 and 3.0±0.5 μM, respectively. The latter were determined using myelin basic protein as the substrate. CaM-kinase II exhibited great differences in the calmodulin requirement for phosphorylation of MAP-2, histone II and myelin basic protein. MAP-2 required the least amount of calmodulin for its phosphorylation. Autophosphorylation of CaM-kinase II resulted in decreased mobility of the α-subunit but apparently not of the β/β′ subunits in sodium dodecyl/sulfate-polyacrylamide gel. Antiserum was raised against the CaM-kinase II α subunit and used for testing cross-reactivity of hen brain enzyme with that of other species. The antiserum which reacted with both α and β subunits of hen brain CaM-kinase II cross-reacted with only the α subunit of rat, mouse, rabbit, cat, dog, pig and human brain samples. The purified hen brain CaM-kinase II is a multifunctional enzyme and resembled rat brain CaM-kinase II several properties. Immunocross-reactivity suggested that there was similarity in the α but not the β/β′ subunits of the hen brain enzyme and the brain enzyme of other species.

Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties

Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for the native enzyme) (Hamada and Kuby, 1978), and the K'd measured for Cr3+ ATP [corrected] and the synthetic peptide I1-45 (Fry et al., 1985b).

The Na +/K +-ATPase reaction of human erythrocytes is not near equilibrium. A 31P-NMR study

We have addressed the question of whether the Na/K+-ATPase in the human erythrocyte is in a state of near-equilibrium by varying the extracellular ratio of Na+ and K+ and following the cytosolic phosphorylation potential by 31P-NMR and by combined enzymatic colorimetric measurements. There was no correlation at room temperature between the extracellular Na+/K+ ratio and the cytosolic phosphorylation potential measured either by NMR or alternative methods. The cytosolic phosphorylation potential measured by NMR was 4100 +/- 1300 (S.E.) M-1 at an extracellular K+ concentration of 5.9 mM (Na+/K+ ratio of 24.3) and 2800 +/- 700 (S.E.) M-1 at 75 mM extracellular K+ (Na+/K+ ratio of 0.99). The chemically determined phosphorylation potential was 6400 +/- 1200 (S.E.) and 5000 +/- 700 (S.E.) M-1 at 5.9 and 75 mM extracellular K+, respectively. Omission of Ca2+ from the buffer solutions did not affect the results. A consistent finding in this study was that the NMR-determined value of ATP was about 10-20% lower than the value determined enzymatically on perchloric acid extracts. The inorganic phosphate (Pi) was fully NMR visible.

Rats with mammary cancer treated with toremifene and interferon: Morphometry and needle aspiration biopsy for determination of ATP and14C-fluorodeoxyglucose content

The combined and separate action of the antiestrogen toremifene (TOR) and recombinant rat gamma interferon (RIF) was studied in rat mammary cancer induced by dimethylbenzanthracene (DMBA). The content of ATP and 14C-fluorodeoxyglucose (FDG) was also determined from fine needle aspiration biopsies (FNAB). RIF alone had no antitumor activity, when measured as the average number of new tumors appearing in RIF and control animals (2.4 vs 2.4 new tumors per animal), while TOR and TOR + RIF had a significant effect (1.2, P less than 0.05 and 0.6, P less than 0.01). Morphometrically, there was a significant decrease in the amount of epithelium in the tumors of the RIF + TOR animals (65% vs 82% in the controls, P less than 0.05); there was conversely an increase in the stromal component (25% vs 14%, NS). It appears that an increase of the stromal compartment is part of the healing process. The feasibility of the FNAB-technique was shown by the finding that there was a close correlation between FDG and ATP content in almost all the groups before and after treatment. Thus, FDG and ATP measure the same phenomenon, i.e., energy content. There was a large variation in the contents of ATP and FDG within and among the groups, which invalidated considerations regarding the predictive value of ATP and FDG content in tumors subject to treatment.

Adenine and pyridine nucleotides in the red blood cells of subjects with solid tumors.

The concentration of adenine (ATP, ADP, AMP) and pyridine (NADP+, NADPH, NAD+, NADH) nucleotides in the erythrocytes of subjects affected by solid tumors was evaluated using a method which allows their simultaneous extraction and reverse-phase high-performance liquid chromatographic analysis. The results showed a lower level of ATP in the erythrocytes of subjects affected by solid tumors, whereas no significant modifications were observed in the other compounds. In fact, the mean value of ATP in these subjects was 27% lower than that of normal adults. This fact is discussed in relation to other enzymatic and metabolic modifications previously observed in red blood cells.

Superoxide generation by pig alveolar macrophages

1. 1. Pig alveolar macrophages generate superoxide anions at a rate of 1.8 nanomoles/1 × 10 6 cells/min. 2. 2. The intracellular value of ATP in resting cells was 4.0 ± 0.1 × 10 −16 mole/cell; in contrast the value in cells generating superoxide anions was 2.0 ± 0.6. 3. 3. Superoxide generation was increasingly inhibited by exposing cells to adenosine from 0.1 to 1.0 mM. 4. 4. Unlike human macrophages, pig cell production of superoxide anions was not inhibited by exposure to the adenosine analog, 2-Cl-adenosine.

Phosphorus-31 nuclear magnetic resonance studies on intact erythrocytes. Determination of intracellular pH and time course changes in phosphorus metabolites.

A method to determine the intracellular pH of intact erythrocytes using phosphorus-31 nuclear magnetic resonance spectroscopy is described. Changes in phosphorus metabolites due to the alkalization of intracellular pH were also examined. The normal erythrocytes gave signals of phosphate groups corresponding to 2,3-bisphosphoglycerate, inorganic phosphate, ATP, and NAD. Among them, the separation between alpha and gamma peaks of ATP was shown to be a good indicator of the intracellular pH free from the perturbation caused by hemoglobin. This method enabled us to determine the intracellular pH of the erythrocytes without any pretreatment. The separation between alpha and gamma peaks of ATP was also dependent on the degree of complexation with Mg2+, and was consistent with approximately 80% of total ATP complexing with Mg2+ in the samples investigated here. The pKa value of ATP in the erythrocytes was estimated to be 6.1 at 23 degrees C, which is lower than the value of 6.5 obtained for the Mg2+-free ATP solution. In the alkalized erythrocytes, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were observed in addition to the metabolites found in the normal erythrocytes. Time course changes in these phosphorus metabolites were followed along with the intracellular pH monitored from ATP peaks.

Interaction of substrates with glutamine synthetase after limited proteolysis

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine triphosphatase activity at the external surface of chicken brain synaptosomes.

An ATP-hydrolysing activity on the external surface of intact synaptosomes from chicken forebrain has been investigated. The observed ATPase activity was not due to leakage of the intracellular ATPase activities, of artefacts resulting from breakage of the nerve endings during the incubation and isolation periods, or to possible contamination by other subcellular particles. Disruption of the synaptosomes resulted in an approximately 2.5-fold increase of the basal, Mg2+-dependent ATPase activity, suggesting that the plasma membrane was acting as permeability barrier to the substrate. ATP hydrolysis was maximal (0.8 mumol Pi/min/mg protein) at pH 8.2 in a medium containing either Mg2+ or Ca2+ ions. Ouabain (0.2 mM) and oligomycin (2 micrograms/mg protein) had no appreciable effect on this ATPase activity. Kinetic studies of the enzyme revealed an apparent Km value of ATP of approximately 4 x 10(-5) M. These data are consistent with the view that the observed ATP hydrolysis was being catalysed by an ectoenzyme, i.e., an enzyme in the plasma membrane of the nerve endings with its active site facing the external medium. The rapid hydrolysis of the released ATP is a suspected function for this ecto-ATPase.

The ATP substrate site of a cyclic-nucleotide-independent protein kinase from porcine liver nuclei.

The ATP substrate site of a second messenger-independent protein kinase of the type NII from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP; they range from 4 microM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP-dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into delta delta values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions. With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its beta-phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N-6 amino group is required in the case of the cAMP-dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N-6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.

Biosynthesis of the Methyl Donors S-adenosylmethionine and S-methylnlethionine in Barley Seedlings (Hordeum vulgare L.)

Summary Methionine adenosyltransferase was partially purified from 2-day-old shoots of barley seedlings. The specific activity of methionine adenosyltransferase was highest in shoots of 2-day-old seedlingsand then the activity rapidly declined. The total activity was also at maximum during germination in the shoots after 4 to 6 days. The specific activity of methionine adenosyltransferase was lower in the roots than in shoots. Barley methionine adenosyltransferase required divalent cations for activity. The greatest activity was found with Mgp2+ or Mn{2+}. K + or NHf4 + increased the enzyme activity. The pH optimum of the enzyme was 8 to 9. The Kfm value of ATP was found to be 0.2 mM, but in the case of methionine non-linear kinetics were obtained, the double reciprocal plots curving downwards. The Kfm value for methionine was estimated to be 0.2 mM. The products in the reaction, S-adenosylmethionine and tripolyphosphate inhibited the enzyme activity. S-adenosylmethionine and tripolyphosphate exhibits non-competitive inhibition patterns versus methionine with Ki values 1.5 mM and 0.1 mM, respectively. S-adenosylmethionine and tripolyphosphate also exhibits non-competitive inhibition patterns versus ATP with Kfi values 1.0 and 0.05 mM, respectively. Methionine S-methyltransferase was partially purified from shoots of 3-day-old barley seedlings. The enzyme does not require Mnp2 + or Znp2+for activity. The Kfm values for methionine and S-adenosylmethionine were both 0.2 mM. The pH optimum for the enzyme activity was 6 to 7. Product inhibition studies showed competitive inhibition between methionine and S-methylmethionine, and between S-adenosylmethionine and S-adenosylhomocysteine with Kfi values 0.3 mM and 0.3 mM, respectively. S-adenosylhomocysteine showed non-competitive inhibition versus methionine with a Kfi of 1.0 mM. The occurrence of methionine S-methyltransferase during germination of the barley seedlings were also investigated, and the enzyme was found in shoot and root tissue and in embryos.

Interaction of adenosine 5′ triphosphate with some dipositive metal ions and isolation and characterization of some novel solid compounds

The isolation and characterization of some dipositive metal ions-ATP complexes have been described. The isolated solid compounds have the composition Na 2M(ATP) nH 2O where n = 2 for M = Ca(II), Mn(II), Fe(II), Co(II), Zn(II) and VO 2+; n = 3 for M = Mg(II), and n = 4 for M = Ni(II), Hg(II) and Pd(II), or Na 2M 3(ATP) 26H 2O where M = Cu(II) and Cd(II), whereas dioxouranium(VI) ion affords NaUO 2(ATPH)2H 2O. The water soluble complexes exhibit 2:1 electrolytic behaviour which is in conformity with the constitutions suggested. The magnetic susceptibilities of the Mn(II), Fe(II), Co(II) and Ni(II) complexes suggest that they have the pseudo-octahedral geometry which fact is supported for Ni(II) and Co(II) compounds by the electronic spectra. The D q value of ATP 4− calculated from the ligand field spectra suggest that its position in the spectrochemical series should be very close to that of H 2O. IR spectra suggest that in most of the cases the metal ions are linked both to the phosphates and to the bases [N(7)] of the adenosine-5′-triphosphate molecule.

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV max,K i andK m values. The normalV max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV max was accompanied by a time-dependent decrease in (K i)ADP, but there was no change in (K m)ATP. The increase inV max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV max was increased by the activating anions by the same factor as theV max in absence of oligomycin. Certain anions, notably azide, decreasedV max, but did not affect (K i)ADP or (K m)ATP. The decrease inV max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria. The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V max)ATP compared with (V max)GTP. The values of (K m)ATP and (K m)GTP were virtually the same. Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.