Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.
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PMID:Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts. 213 61

The functional properties of mitochondria bound hexokinase are compared in two subpopulations of the HT29 human colon cancer cell-line: (1) the HT29 Glc+ cells, cultured in the presence of glucose, which are poorly differentiated and highly glycolytic and (2) the HT29 Glc- cells, adapted to grow in a glucose-free medium, which are 'enterocyte-like' differentiated and less glycolytic when given glucose (Zweibaum et al. (1985) J. Cell Physiol. 122, 21-28). The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase are found to be twice as high in Glc+ cells when compared to Glc- cells. Besides, the respiration rate is decreased in Glc+ cells compared to Glc- cells. These results correlate with the higher glycolytic rate in Glc+ cells. In many tissues, it has been shown that the binding of hexokinase to the mitochondrial outer membrane allows a preferential utilization of the ATP generated by oxidative phosphorylation which, in turn, is activated by immediate restitution of ADP. In highly glycolytic cancer cells, although a large fraction of hexokinase is bound to the mitochondria, the existence of such a channeling of nucleotides is still poorly documented. The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from both Glc+ and Glc- cells either with exogenous ATP or with ATP generated by mitochondria supplied with ADP and succinate (endogenous ATP). Diadenosine pentaphosphate (Ado2P5), oligomycin and carboxyatractyloside (CAT) were used in combination or separately as metabolic inhibitors of adenylate kinase, ATP synthase and ATP/ADP translocator, respectively. Exogenous ATP appears to be 6.5-times more efficient than endogenous ATP in supporting hexokinase activity in the mitochondria from Glc+ cells and only 1.8-times cells. The rate of oxidative phosphorylation being higher in mitochondria from Glc- cells, hexokinase activity is higher in this model when ATP is generated by respiration. Furthermore, in Glc+ mitochondria, the adenylate kinase reaction appears to be an important source of endogenous ATP for bound hexokinase, while, in Glc- mitochondria, hexokinase activity is almost totally dependent on the ATP generated by oxidative phosphorylation. This result might be explained by our previous finding that mitochondria from Glc+ cells lack contact sites between outer and inner membrane, whereas numerous contacts were observed in mitochondria from Glc- cells (Denis-Pouxviel et al. (1987) Biochim. Biophys. Acta 902, 335-348).
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PMID:Study on ATP-generating system and related hexokinase activity in mitochondria isolated from undifferentiated or differentiated HT29 adenocarcinoma cells. 252 30

A mathematical procedure is presented which permits to calculate the steady-state concentrations of AMP, ADP and ATP in an ATP-regenerating assay containing pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. The accuracy of this procedure is demonstrated by the agreement of the calculated concentrations with the experimental data obtained in measurements of mitochondrial ATPase activities. The computer-assisted procedure can be employed (a) to determine extremely low adenine nucleotide concentrations which are difficult to obtain by direct measurements and (b) to adjust and to optimize the assay conditions according to the specific requirements of the experiment, including high concentrations of ATP and prescribed ATP/ADP ratios.
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PMID:A computer assisted method to control the steady state of an ATP-regenerating assay. 275 34

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.
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PMID:Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides. 289 74

Previous studies from this laboratory have shown that mitochondrial bound hexokinase is markedly elevated in highly glycolytic hepatoma cells (Parry, D. M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). A pore-forming protein, porin, within the outer membrane appears to comprise at least part of the receptor site (Nakashima, R.A., Mangan, P.S., Colombini, M., and Pedersen, P.L. (1986). Biochemistry 25, 1015-1021). In studies reported here experiments were carried out to assess the functional significance of mitochondrial bound tumor hexokinase. Two approaches were used to determine whether the bound enzyme has preferred access to mitochondrially generated ATP relative to cytosolic ATP. The first approach compared the time course of glucose 6-phosphate formation by AS-30D hepatoma mitochondria under conditions where ATP was regenerated endogenously via oxidative phosphorylation or exogenously by added pyruvate kinase and phosphoenolpyruvate. The second approach involved the measurement of the specific radioactivity of glucose 6-phosphate formed following the addition of [gamma-32P]ATP to either phosphorylating or nonphosphorylating AS-30D mitochondria. Both approaches provided results which show that the source of ATP for bound hexokinase is derived preferentially from the ATP synthase residing within the inner mitochondrial membrane compartment rather than from the medium (i.e. from the cytosolic compartment). These results provide the first direct demonstration that the exceptionally high level of hexokinase bound to mitochondria of highly glycolytic tumor cells has preferred access to mitochondrially generated ATP, a finding that may have rather profound metabolic significance for such tumors.
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PMID:Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP. 318 54

1. The initial rapid phase of ATP hydrolysis by bovine heart submitochondrial particles or by soluble F1-ATPase is insensitive to anion activation (sulphite) or inhibition (azide). 2. The second slow phase of ATP hydrolysis is hyperbolically inhibited by azide (Ki approximately 10(-5) M); the inosine triphosphatase activity of submitochondrial particles or F1-ATPase is insensitive to azide or sulphite. 3. The rate of interconversion between rapid azide-insensitive and slow azide-sensitive phases of ATP hydrolysis does not depend on azide concentration, but strongly depends on ATP concentration. 4. Sulphite prevents the interconversion of the rapid initial phase of the reaction into the slower second phase, and also prevents and slowly reverses the inhibition by azide. 5. The presence of sulphite in the mixture when ADP reacts with ATPase of submitochondrial particles changes the pattern of the following activation process. 6. Azide blocks the activation of ATP-inhibited ATPase of submitochondrial particles by phosphoenolpyruvate and pyruvate kinase. 7. The results obtained suggest that the inhibiting effect of azide on mitochondrial ATPase is due to stabilization of inactive E*.ADP complex formed during ATP hydrolysis; the activation of ATPase by sulphite is also realized through the equilibrium between intermediate active E.ADP complex and inactive E*.ADP complex.
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PMID:Kinetic mechanism of mitochondrial adenosine triphosphatase. Inhibition by azide and activation by sulphite. 621 Nov 71

1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.
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PMID:Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics. 621 Nov 73

A method to optimize enzymatic assays by using pyruvate kinase and lactate dehydrogenase enzymes is presented and applied to mitochondrial ATPase as an example. Optimum amounts of auxiliary enzymes, to obtain either a 99% of the initial rate in a given time (t99) or a given lag period (L), are calculated from their apparent Michaelis constants (Kapp) in the medium used and their prices per enzymatic international unit.
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PMID:Enzymatic assays: optimization of systems by using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. 621 43

The 2-azidoadenine nucleotides show promise as photoaffinity probes. Substitution at the C-2 position should favor an anti conformation and enable binding of the analogue to enzyme sites which exhibit low affinity for the 8-azidoadenine derivatives. The 2-azidoadenine nucleotides were found to be substrates for pyruvate kinase, phosphofructokinase, adenylate kinase, hexokinase and the mitochondrial F1-ATPase. However, tautomerism of 2-azidoadenine nucleotides to two nonphotoreactive tetrazole forms complicates kinetic analyses and their use as photoaffinity probes. An analysis of the ultraviolet spectra of these analogues enables an estimation of the tetrazolo isomer content and the rates of tautomerization. The photoreactive azido isomer was found to represent only 45% of the total analogue population in neutral aqueous solution. The azidoazomethine-tetrazole equilibrium favors the azido isomer in acidic or nonpolar solutions. The first-order rate constants at 25 degrees C were determined to be 0.017 min-1 and 0.021 min-1 for tautomerism to the azido and tetrazolo isomers, respectively. Prior equilibration of the probe in various solvents thus allows investigation of the analogue's behavior with an enzyme system at different, essentially fixed, isomer ratios. The determination of the impact of the tetrazolo tautomers on the system allows optimization of conditions for photoaffinity-labeling experiments.
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PMID:Tautomerism of 2-azidoadenine nucleotides. Effects on enzyme kinetics and photoaffinity labeling. 633 19

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.
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PMID:The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein. 644 68


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