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1.
The oxygen dependence of cellular energy metabolism. 总被引:14,自引:0,他引:14
D F Wilson M Erecińska C Drown I A Silver 《Archives of biochemistry and biophysics》1979,195(2):485-493
Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μm with an apparent Km for oxygen of less than 1 μm. In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μm downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic and intramitochondrial because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and . 相似文献
2.
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
3.
The ATP/ADP exchange is shown to be a partial reaction of the by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to , -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration () required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+. 相似文献
4.
Efficiencies of oxidative phosphorylation ( ratios), intracellular high-energy phosphate pools (ATP and ADP) under aerobic and anaerobic dark conditions, and photosynthetic oxygen evolution measured with intact cells of Anacystis nidulans were found to be specifically depressed by NaCl, but not by KCl. A scheme is proposed which explains the deleterious effect of sodium on the energy metabolism of A. nidulans by competition for protons between ATP synthesis and active sodium extrusion. 相似文献
5.
The role of acetaldehyde (AcH) in the ethanol-induced shift toward reduction of the cytosolic and mitochondrial free NAD+/free NADH ratios and its effect on the phosphorylation potential was investigated in livers of fed, intact rats given ethanol (1 g/kg ip). Calcium cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, was administered to block predominantly intramitochondrial NADH production from AcH oxidation. Compared with ethanol alone, cyanamide almost totally reversed the elevation of the β-OH-butyrate/acetoacetate ratio but only slightly reduced the lactate/ pyruvate ratio, which was calculated to be in near equilibrium with the hepatic ethanol/ AcH ratio after cyanamide. Ethanol or cyanamide alone had no effect on ATP, ADP, or Pi, but together they significantly decreased the ratio by increasing both ADP and Pi levels. No association between changes in the phosphorylation potential and the redox states was, however, observed. An ethanol-induced increase in AMP was abolished by cyanamide. The results demonstrate that the effect of ethanol on the mitochondrial redox state requires active AcH oxidation and suggest that moderate AcH accumulation likely to occur during alcohol-aversive drug treatment significantly lowers the cellular phosphorylation potential. 相似文献
6.
A.L. Moore C. Jackson B. Halliwell J.E. Dench D.O. Hall 《Biochemical and biophysical research communications》1977,78(2):483-491
Intact spinach leaf mitochondria are capable of oxidising glycine with good respiratory control and the oxidation is coupled to 3 phosphorylation sites. The intramitochondrial localisation of glycine decarboxyllation has been studied and it is demonstrated that the enzyme system is associated with the inner membrane of spinach leaf mitochondria. Both glycine decarboxylation and glycine dependent O2 uptake are stimulated by ADP and FCCP and are sensitive to electron transport inhibitors. Both processes showed no requirements for co-factors. We suggest that glycine decarboxylase is coupled to the electron transport chain an NAD+-linked system and that during rapid photorespiration glycine oxidation synthesises considerable amounts of ATP outside of the chloroplast. 相似文献
7.
Rat heart mitochondria oxidizing pyruvate (in the presence of 20% as much malate) took up nearly the amount of oxygen required for complete oxidation to CO2. Thus pyruvate, a physiological substrate of the citrate cycle, is oxidized through the entire cycle in these mitochondria, and they seem suitable for study of regulation of integrated mitochondrial energy transduction. By addition of graded amounts of hexokinase or pyruvate kinase to the suspending medium (in the presence of excess glucose or phosphoenolpyruvate), a wide range of steady-state values of the concentration ratio was obtained. At a constant concentration of phosphate, the steady-state rate of oxygen uptake by rat heart mitochondria oxidizing pyruvate was a function of the adenylate energy charge or of the ratio, and relatively independent of the absolute concentrations of these nucleotides. The oxygen uptake rates typically spanned a range of about 20-fold. At very high values of the ratio, the rate of oxygen uptake is much lower than the “state 4” rate seen after added ADP has been phosphorylated. This result suggests that “state 4” respiration, at least in these freshly prepared mitochondria, measures the rate at which ADP is made available by ATPase activity, rather than indicating uncoupling of electron transport from phosphorylation. The concentration of orthophosphate affected the rate of oxygen uptake and the pattern of response to the ratio or the energy charge, but the effects did not seem interpretable in terms of the mass-action expression for hydrolysis of ATP, (Pi. 相似文献
8.
Relations between extramitochondrial and intramitochondrial adenine nucleotide systems 总被引:1,自引:0,他引:1
Wolfgang Kunz Ralf Bohnensack Günter Böhme Uwe Küster Gerold Letko Peter Schönfeld 《Archives of biochemistry and biophysics》1981,209(1):219-229
The control of oxidative phosphorylation by the extramitochondrial ratio and [Pi] was investigated by incubations of isolated mitochondria with an ADP regenerating system and by a new perifusion technique using glass filters for immobilization of mitochondria. With mitochondria from different sources oxidizing different substrates and with both techniques, similar results were obtained. Changes of the extramitochondrial ratio from about 100 to 5 transfer mitochondria from the resting state (state 4) to the fully active state (state 3). The importance of the adenine nucleotide translocator in this transition was demonstrated by the influence of its specific inhibitor carboxyatractyloside. The sensitivity to the inhibitor was more pronounced in states with high ratios than in the fully active state. In the hexokinase-glucose system the action of the inhibitor caused a transition to a new steady state, where a decreased ratio overcomes the inhibition. Thus, a partial inhibition of the translocator shifted the control characteristics to lower ratios. When the concentration of inorganic phosphate was decreased, the main effect was a reduction of the maximum rate of oxidative phosphorylation (i.e., in state 3), whereas the sensitive range was not altered. This effect is caused by changes in the intramitochondrial phosphorylation potential. Furthermore, this indicates that the kinetic properties of the adenine nucleotide translocator prevent a simple equilibration of the phosphorylation potential across the inner membrane. This is also demonstrated by the fact that the extramitochondrial formation of glucose-6-phosphate and the intramitochondrial synthesis of citrulline compete for ATP. 相似文献
9.
M S Balakrishnan T R Sharp J J Villafranca 《Biochemical and biophysical research communications》1978,85(3):991-998
Oxygen-18 exchange out of [18O]Pi catalyzed by Mg2+-activated unadenylated glutamine synthetase from was followed by 31P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the in the species P18O4, P18O316O1, P18O216O2, P18O116O3, P16O4 during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of Pi. The rate constant for exchange was 410±40 min?1 while the rate constant for net reaction (ATP formation) was 62±4 min?1. Thus exchange proceeds ~7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ( (1976) , 5558) for the Mn2+-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) , 476) was successfully used to fit the oxygen exchange data in this paper. 相似文献
10.
The steady state levels of intermediary metabolites were measured in freeze clamped, developing, dormant, and activated infective Ascaris lumbricoides eggs. The ratio is low in the developmental stages and rises sharply in the dormant egg; on activation of the dormant egg the ratio falls. The levels of the phosphorylated glycolytic intermediates of acetyl-CoA and of isocitrate do not change markedly during development, but the levels of lactate, citrate, 2-oxoglutarate, glutamate, succinate, and malate all show significant changes in the developing, dormant, and activated egg. The dormant egg also appears to be characterized by a low cytoplasmic redox potential. 相似文献
11.
Isolated hepatocytes incubated with 2 mm ornithine-10 mm glutamine as substrates and challenged with either glucagon, epinephrine, or phenylephrine exhibited stimulated rates of urea production, and mitochondria isolated from these cells displayed an increased rate of energy-dependent citrulline formation. There was no change in the total carbamyl phosphate synthetase I activity, nor mitochondrial content of the positive effector N-acetyl glutamate after acute hormonal treatment. The time of onset of ureogenesis and its sensitivity to glucagon were compared with stimulation of glucose production from lactate-pyruvate. No apparent differences in time of onset or sensitivity of the responses were observed indicating both pathways may be stimulated by a common mechanism. Mitochondria prepared from cells treated with catecholamines exhibited increased rates of State 3 respiration and increased uncoupler-dependent ATPase activity, in addition to the increased rates of citrulline formation. There was also an elevated intramitochondrial content of ATP and an increased ratio. The catecholamine-induced stimulation of ureogenesis was mediated by an α-adrenergic cyclic AMP independent mechanism. The addition of the α-adrenergic antagonist, dihydroergotamine, blocked both the epinephrine-induced stimulation of ureogenesis and also the stimulated functions in the isolated mitochondria. dl-Propranolol, a β-antagonist, inhibited the rise in cyclic AMP due to epinephrine, but had no effect on any of the other reactions measured. The effects of catecholamines on citrulline formation and urea production are correlated with the increased capacity of the mitochondria to generate ATP. It is suggested that both glucagon and catecholamines, acting via independent mechanisms, stimulate electron transport and the activity of the ATP-forming enzyme complex. The consequent elevated intramitochondrial ATP levels and ratio enhance the rate of citrulline formation and hence ureogenesis. 相似文献
12.
(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) Vm for ADP-arsenate synthesis was about 2-times lower than Vm for ATP-synthesis. With saturating [ADP], K(Asi) was about 20% higher than K(Pi). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of vs. were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased ratio. High concentrations of the fixed substrate cause a similar increase in , but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (Asi) stimulate ATP synthesis supported by low concentrations of Pi and ADP about 2-fold. (5) At high ADP concentrations, the apparent Ki of Asi for inhibition of ATP-synthesis was 2–3-times higher than the apparent Km of Asi for arsenylation; the apparent Ki of Pi for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent Km of Pi for ATP synthesis. (6) The results are discussed in terms of a model in which Pi and Asi compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion. 相似文献
13.
Albert K. Groen Richard C. Vervoorn Ron J.A. Wanders Roelof Van der Meer Joseph M. Tager 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(2):172-177
The cytosolic phosphate potential was estimated in isolated rat liver parenchymal cells incubated with various gluconeogenic substrates. The value of the cytosolic ratio was either estimated directly from measurements of ATP, ADP and Pi after digitonin fractionation of the cells, or calculated by the metabolite indicator method. When cells were incubated with lactate, pyruvate or alanine so that net flux through the indicator enzymes was in the gluconeogenic direction, there was excellent agreement between the values obtained by the two methods over a wide range of fluxes. However, when the cells were incubated with substrates that could be converted both to glucose and to lactate so that net flux through the indicator enzymes was in the glycolytic direction, a large difference in the values of the cytosolic ) ratio as derived by the two methods was observed. It is concluded that the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase plus 3-phosphoglycerate kinase is out of equilibrium when flux through the reaction is in the glycolytic direction, and that use of the metabolite indicator method for the calculation of the cytosolic phosphate potential under these conditions leads to erroneous values. 相似文献
14.
Thiol modulation of the chloroplast protonmotive ATPase (CF0-CF1) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH4Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF0-CF1 increased the observed Michaelis constant for ADP (Km(ADP)) and the apparent maximum rate (Vapp of photophosphorylation by the same factor, so that ratio was not altered. was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF0-CF1 is presented to explain the dependence of α on ΔpH. Thiol modulation of CF0-CF1 is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process. 相似文献
15.
The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10?5m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10?4m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The ratio increases and tends to level off as the incubation time increases. No increase in ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process. 相似文献
16.
A M Requero J P Díaz M S Ayuso-Parrilla R Parrilla 《Archives of biochemistry and biophysics》1979,195(1):223-234
The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD+ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the ratio brought about by glucagon may be secondary to the increase in the ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the and ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD+ system. 相似文献
17.
P V Blair 《Biochemical and biophysical research communications》1979,88(2):537-544
ATP or combinations of ATP with EDTA and EGTA can act as chelators to support succinate-driven, phosphate-requiring expansion of mitochondrial inner membrane-matrices. Contraction of these swollen mitochondria can be induced with antimycin, MgCl2 and ADP. The magnitude of ADP-induced contraction of mitochondria, swollen in the presence of ATP, is dependent on [ADP] and may be altered by the extramitochondrial concentrations of both Pi and ATP. In fact, the extent of contraction (+ΔA520) is a linear function of the thermodynamic parameter, ?ΔGp (free energy of hydrolysis of ATP), provided excessive concentrations of reactants are not present and the extents of matrix swelling are similar ( is about 0.250) before starting contraction with ADP. 相似文献
18.
The total adenine nucleotide content of rat liver mitochondria was varied in vitro over a wide range in order to investigate a possible relationship between net changes in the total matrix ATP + ADP + AMP content and the overall rate of citrulline synthesis. Isolated mitochondria were specifically depleted of matrix adenine nucleotides by incubating with inorganic pyrophosphate (G. K. Asimakis and J. R. Aprille, 1980, Arch. Biochem. Biophys.203, 307–316); alternatively, matrix adenine nucleotides were increased by incubating mitochondria with 1 mm ATP at 30 °C. No exogenous ATP or ADP was included in the subsequent incubations for the determination of citrulline synthesis. Rates varied from 0.1 to 1.6 μmol citrulline/mg protein/h as a linear function of total adenine nucleotide content in the range 2–15 nmol (ATP + ADP + AMP)/mg protein. Further increases in the matrix ATP + ADP + AMP content caused no further increase in citrulline synthesis rates. Changes in the total adenine nucleotide content were reflected in proportional changes in both the ATP and ADP content of the matrix. The ratio did not change significantly. Therefore, the variations in citrulline synthesis were most simply explained as the effect of different concentrations of ATP on the activity of carbamoyl-phosphate synthetase. It was concluded that net changes in the total adenine nucleotide content can contribute to the control of citrulline synthesis. These findings are significant in the context of recent evidence which shows that the matrix adenine nucleotide pool size is under hormonal control. 相似文献
19.
The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin 总被引:3,自引:0,他引:3
Evidence is presented that both myosin and actomyosin in presence of Mg2+ and KCl catalyze an incorporation of 32Pi into ATP. The rate with actomyosin is about the rate of ATP hydrolysis; the rate with myosin is less than of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of 32Pi incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [32P]-ATP present and the rate constant, k?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔGo for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔGo for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage. 相似文献
20.
The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca2+, Mg2+-activated ATPase of . In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an mutant of was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked. 相似文献