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1.
2.
A biochemical assay for the measurement of ATP synthesis coupled to electron transport in the presence of adenylate kinase was developed as an alternative to using the conventional Clark-type oxygen electrode. The assay utilizes P1,P5-di-(adenosine-5′)-pentaphosphate which is shown to be a competitive inhibitor with MgADP for rat liver mitochondrial adenylate kinase (Ki = 7.04 × 10?8m) and was found to have no effect on oxidative phosphorylation of either intact mitochondria or submitochondrial particles. 相似文献
3.
Robert A. Mitchell John A. Russo Candace M. Lamos 《Journal of cellular biochemistry》1975,3(3):256-260
1,N6-Ethenoadenosine diphosphate (ϵ-ADP) inhibits reverse electron flow (succinate → NAD+ driven by ATP) by competing with ATP, in contrast to ADP which we have shown previously to be a noncompetitive inhibitor. From these and other data it is concluded that the noncompetitive inhibition noted with ADP results from a combination of competitive inhibition plus non- or uncompetitive inhibition, the former occuring at a relatively nonspecific catalytic site and the latter at an extracatalytic site apparently quite specific for ADP. ADP, which stimulates ATP ⇌ H2O and Pi ⇌ H2O exchanges appears to be necessary for inhibition by arsenate of these exchanges. It is suggested that the ATP-supported Pi ⇌ H2O exchange may be predominantly of the medium or intermediate type, depending on the concentrations of the Mg2+ complexes of ADP and Pi. Thus only exchanges involving medium ADP and Pi would be expected to show arsenate sensitivity. 相似文献
4.
Ceil A. Herman Terry V. Zenser Bernard B. Davis 《Prostaglandins & other lipid mediators》1977,14(4):679-687
Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluated for their effects on PGE2 production and ATP levels. None of the inhibitors affected PGE2 synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE2 and ATP levels. This suggest that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather an effect on oxidative phosphorylation.When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was ommitted, PGE2 levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE2 levels, with a maximal effect at 10mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis.These results indicate that oxygen (substrate) availability can limit inner medullary PGE2. In view of the low pO2 in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin productin in this tissue. 相似文献
5.
Dominique Freeman Sylvia Bartlett George Radda Brian Ross 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(2):325-336
31P-NMR has been used to quantify inorganic phosphate (Pi) and high-energy phosphates in the isolated, functioning perfused rat kidney, while monitoring oxygen consumption, glomerular filtration rate and sodium reabsorption. Compared with enzymatic analysis, 100% of ATP, but only 25% of ADP and 27% of Pi are visible to NMR. This is indicative that a large proportion of both ADP and Pi are bound in the intact kidney. NMR is measuring free, and therefore probably cytosolic concentrations of these metabolites. ATP synthesis rate, measured by saturation transfer NMR shows the P:O ratio of 2.45 for the intact kidney. This is close to the theoretical value, suggesting the NMR visible pool is that which is involved in oxidative phosphorylation. The energy cost of Na transport, calculated from the theoretical Na:ATP of 3.0 exceeded the measured rate of ATP synthesis. Instead, Na:ATP for active transport in the perfused kidney was 12. Since the phosphorylation potential () by NMR was 10 000 M?1, the free-energy of ATP hydrolysis was 52 kJ/mol. Using this figure, the rate of ATP hydrolysis observed could fully account for the observed rate of sodium reabsorption. 相似文献
6.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1980,597(1):100-111
can be phosphorylated by its substrate ATP as well as by its product inorganic phosphate. The maximal capacity for phosphorylation by either of these two substances is one mol phosphate per mol enzyme. In order to investigate whether the enzyme molecule possesses only one phosphorylation site common to ATP and Pi, or two phosphorylation sites, one for ATP and one for Pi, dual phosphorylation of the enzyme has been carried out. Under conditions, which are maximally favourable for each type of phosphorylation, successive phosphorylation by Pi and ATP leads to a maximal incorporation of only one mol phosphate per mol enzyme. The phosphorylation capacity for ATP decreases by the same amount as the Pi-phosphorylation level increases, without an effect on the apparent affinity for ATP.The results can be explained by assuming either a single common phosphorylation site for Pi and ATP, or a conformational change of the enzyme following phosphorylation by Pi, which excludes phosphorylation by ATP. 相似文献
7.
We examined the importance of tyrosine kinase(s) on the ATP-evoked Ca2+ entry and DNA synthesis of thyroid FRTL-5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72-kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca2+ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca2+, but the capacitative Ca2+ or Ba2+ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca2+ ([Ca2+]i), whereas the Ba2+ entry was not increased. Phorbol 12-myristate 13-acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP-evoked phosphorylation of MAP kinase and attenuated both the ATP- and the PMA-evoked DNA synthesis. However, genistein did not inhibit the ATP-evoked expression of c-fos. Furthermore, genistein enhanced the ATP-evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca2+-dependent mechanism. A genistein-sensitive mechanism participates, in part, in the ATP-evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca2+ entry in FRTL-5 cells. J. Cell. Physiol. 175:211–219, 1998. © 1998 Wiley-Liss, Inc. 相似文献
8.
Richard L. Cross 《Journal of bioenergetics and biomembranes》1988,20(4):395-405
Recent structural and kinetic studies of F1 and F0F1 are reviewed with regard to their implications for the binding change mechanism for ATP synthesis by oxidative phosphorylation and photophosphorylation. It is concluded that at least two and probably all three of the catalytic sites on F1 are functionally equivalent despite permanent structural asymmetry in the soluble enzyme. A rotary mechanism in which all three catalytic subunits experience all possible interactions with the single-copy subunits during turnover is thought not to apply to soluble F1 but remains an attractive model for the membrane bound enzyme. 相似文献
9.
It has proposed that hexokinase bound to mitochondria occupies a preferred site to wich ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740–749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) ot any combination of these, suggesting a source of ATP in addition to oxidative phosphorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentraions, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed. 相似文献
10.
J.F. Allen 《FEBS letters》1984,166(2):237-244
Protein phosphorylation in isolated, intact pea chloroplasts was measured during the onset of CO2-dependent O2 evolution. Total incorporation of 32P (from 32Pi) into the light-harvesting chlorophyll a/b—protein was found to be less sensitive than O2 evolution to inhibition by the uncouplers FCCP and NH4C1 It is concluded that changes in the rate of ATP synthesis cannot affect protein phosphorylation without also affecting the rate of CO2-fixation in this system. The ATP/ADP ratio is therefore unlikely to regulate photosynthetic protein phosphorylation under normal physiology conditions. 相似文献
11.
This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and Pi and for the release of ATP bound at the catalytic sites of F1-ATPase. The process of ATP synthesis on the surface of F1 from F1-bound ADP and Pi appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a lowK
m
(for ADP and Pi)-lowV
max mode and a highK
m
-highV
max mode. TheK
m
extremes for ADP are 2–3 µM and 120–150 µM, andV
max
for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 s–1 (mole F1)–1 at 30°C, which corresponds to 11 µmol ATP (min · mg of protein)–1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunitc) of the membrane sector (F0) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F1, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F1 in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential () and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions is exquisitely sensitive to changes in the rates of energy production and consumption. 相似文献
12.
Summary Control of mitochondrial respiration depends on ADP availability to the F1ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · Pi which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca2+ concentrations. The Ca2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca2+ by activation of Ca2+-sensitive dehydrogenases. The Ca2+-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities. 相似文献
13.
Changes in respiratory activity, transmembrane electric potential, and ATP synthesis as induced by additions of limited amounts of ADP and Pi to tightly coupled inverted (inside-out) Paracoccus denitrificans plasma membrane vesicles were traced. The pattern of the changes was qualitatively the same as those observed for coupled mitochondria during the classical State 4-State 3-State 4 transition. Bacterial vesicles devoid of energy-dependent permeability barriers for the substrates of oxidation and phosphorylation were used as a simple experimental model to investigate two possible mechanisms of respiratory control: (i) in State 4 phosphoryl transfer potential (ATP/ADP × Pi) is equilibrated with proton-motive force by reversibly operating F1·Fo-ATPase (thermodynamic control); (ii) in State 4 apparent “equilibrium” is reached by unidirectional operation of proton motive force-activated F1·Fo-ATP synthase. The data support the kinetic mechanism of the respiratory control phenomenon. 相似文献
14.
Liguzinski P Korzeniewski B 《American journal of physiology. Cell physiology》2006,291(6):C1213-C1224
It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction 相似文献
15.
Yakov M. Milgrom 《BBA》2010,1797(10):1768-1774
The effect of inorganic phosphate (Pi) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F1-ATPase from beef heart mitochondria (ndMF1) has been investigated. It is shown for the first time that Pi decreases the apparent rate constant of uni-site ATP binding by ndMF1 3-fold with the Kd of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, Pi also shifts equilibrium between bound ATP and ADP + Pi in the direction of ATP synthesis with the Kd of 0.17 ± 0.03 mM. However, 10 mM Pi does not significantly affect ATP binding during multi-site catalysis. 相似文献
16.
A phosphorylation system for formation of ATP from AMP by Zymolyase-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was developed as an ATP production process. This system was shown to be an energy conversion system, from a reduced C1 -compound to ATP through reduction of NAD+ and oxidative phosphorylation but not substrate level phosphorylation, together with phosphorylation of AMP to ADP.Reaction conditions for the ATP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pressure, and oxygen supply. Under the optimal conditions, 26 mM (13 g/liter) and 8.5 dim (4g/liter) of ATP were produced with methanol and formate as C1 -substrate, respectively. 相似文献
17.
O. Bârzu F. Eckstein S. Dancea I. Petrescu C. Tărmure L.D. Ngoc A. Hodârnău H.H. Mantsch 《BBA》1979,547(2):361-369
Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria. 相似文献
18.
Blood respiratory properties of rainbow trout,Salmo gairdneri: Responses to hypoxia acclimation and anoxic incubation of blood in vitro 总被引:1,自引:1,他引:0
Vilhelm Tetens Gunnar Lykkeboe 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1981,145(1):117-125
Summary Blood respiratory properties of rainbow trout were determined following acclimation to normoxia and two levels of hypoxia.The most prominent response appeared to be an increase in blood O2 affinity graded to the level of hypoxia. TheP
50 values (at pH 7.8 and 20°C) were 24.1 21.7 and 16.8 mm Hg when specimens were acclimated to water O2 tensions of 150, 80 and 50 mm Hg, respectively. The blood O2 affinity was closely correlated with the erythrocytic ATP concentration. The stepwise correlation of ATP andP
50, when trout were exposed to graded oxygen lack in the water, indicates that the blood O2 affinity is precisely regulated.Anoxic incubation of trout blood in vitro induced a rapid reduction in erythrocytic ATP concentration (t
1/2=75 min), which was closely correlated to theP
50 value. The drop inP
50 value during anoxic exposure can be explained partly by the direct allosteric effect of a decreased erythrocytic ATP concentration and partly by the modified Donnan distribution of protons across the red cell membrane. Reoxygenation of the incubated blood, however, only partly re-established the erythrocytic ATP concentration, with a concurrent rise inP
50 value.The results invite discussion about the mechanism, by which fish regulate their blood O2 affinity. It is concluded, that it is regulated at the organismal rather than at the red cell level.Abbreviation (E)
erythrocytes, erythrocytic 相似文献
19.
Kalyan?C. Vinnakota Jason?N. Bazil Fran?oise Van?den?Bergh Robert?W. Wiseman Daniel?A. Beard 《Biophysical journal》2016,110(4):972-980
To determine how oxidative ATP synthesis is regulated in the heart, the responses of cardiac mitochondria oxidizing pyruvate to alterations in [ATP], [ADP], and inorganic phosphate ([Pi]) were characterized over a range of steady-state levels of extramitochondrial [ATP], [ADP], and [Pi]. Evolution of the steady states of the measured variables with the flux of respiration shows that: (1) a higher phosphorylation potential is achieved by mitochondria at higher [Pi] for a given flux of respiration; (2) the time hierarchy of oxidative phosphorylation is given by phosphorylation subsystem, electron transport chain, and substrate dehydrogenation subsystems listed in increasing order of their response times; (3) the matrix ATP hydrolysis mass action ratio [ADP] × [Pi]/[ATP] provides feedback to the substrate dehydrogenation flux over the entire range of respiratory flux examined in this study; and finally, (4) contrary to previous models of regulation of oxidative phosphorylation, [Pi] does not modulate the activity of complex III. 相似文献
20.
Kathryn M. Johnson Lara Swenson Anthony W. Opipari Jr. Rolf Reuter Nawid Zarrabi Carol A. Fierke Michael Börsch Gary D. Glick 《Biopolymers》2009,91(10):830-840
The mitochondrial F1Fo‐ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F1Fo‐ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F1 domain and inhibits F1Fo‐ATPase‐catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F1Fo‐ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower Ki values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F1Fo‐ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single‐molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F1Fo‐ATPase catalyzes ATP synthesis and hydrolysis. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 830–840, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献