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1.
The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by (31)P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, F(f) = PCr --> ATP, and reverse flux, F(r) = ATP --> PCr = 3.3 mm.s(-1)), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (F(f) = 1.7 x F(r)), and (iii) a mitochondrial CK displaced toward PCr synthesis (F(f) = 0.3 and F(r) = 2.6 mm.s(-1)). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed.  相似文献   

2.
How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes (myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR. To get insight in the energy transfer pathway in the perfused rat heart, we developed a combined analysis of several protocols of magnetization transfer associated with biochemical data and quantitatively evaluated which scheme of energetic exchange best describes the NMR data. This allows to show the kinetic compartmentation of subcellular CKs and to quantify their fluxes. Interestingly, we could show that the energy transfer pathway shifts from the phosphocreatine shuttle in the oxygenated perfused heart to a direct ATP diffusion from mitochondria to cytosol under moderate inhibition of ATP synthesis. Furthermore using NMR measured fluxes and the known kinetic properties of the enzymes, it is possible to model the system, estimate local ADP concentrations and propose hypothesis for the versatility of energy transfer pathway. In the normoxic heart, a 3-fold ADP gradient was found between mitochondrial intermembrane space, cytosol and ADP in the vicinity of ATPases. The shift from PCr to ATP transport observed when ATP synthesis decreases might result from a balance in the activity of two populations of ANT, either coupled or uncoupled to CK. We believe this NMR approach could be a valuable tool to reinvestigate the control of respiration by ADP in the whole heart reconciling the biochemical knowledge of mitochondrial obtained in vitro or in skinned fibers with data on the whole heart as well as to identify the implication of bioenergetics in the pathological heart.  相似文献   

3.
Forward [creatine phosphate (CP)----adenosine 5'-triphosphate (ATP)] and reverse (ATP----CP) fluxes of myocardial creatine kinase (CK) measured by using 31P nuclear magnetic resonance (NMR) and conventional saturation transfer (CST) methods are unequal; this is a paradoxical result because during steady state fluxes into and out of the CP pool must be the same. These measurements, however, treat the CK reaction as a two-site exchange problem and ignore the presence of the ATP gamma in equilibrium Pi exchange involving the ATPases. We have applied a method [U?urbil, K. (1985) J. Magn. Reson. 64, 207] based on the saturation of multiple resonances, by which a single unidirectional rate constant can be measured unequivocally in the presence of multiple exchanges, to the measurement of CK fluxes in isovolumic rat hearts perfused under three different conditions; two of the three perfusion conditions showed a large discrepancy in the CK fluxes determined by CST, and one did not. In contrast, when the effect of the ATP gamma in equilibrium Pi exchange on the CK rate measurements was eliminated, multiple saturation transfer (MST) measurements on the same hearts yielded equal forward and reverse fluxes in all cases. The rate constant for the ATP gamma----CP conversion measured by MST was larger than the value obtained by the conventional methodology whereas both methods gave the same rate constant in the CP----ATP direction. These results demonstrate that the cause of the paradoxical data obtained by CST measurements of CK kinetics is the ATP gamma in equilibrium Pi exchange and that CK rates when determined rigorously are consistent with the CK reaction being in equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
A challenge in the understanding of creatine kinase (CK) fluxes reflected by NMR magnetization transfer in the perfused rat heart is the choice of a kinetic model of analysis. The complexity of the energetic pathways, due to the presence of adenosine triphosphate (ATP)-inorganic phosphate (Pi) exchange, of metabolite compartmentation and of subcellular localization of CK isozymes cannot be resolve from the sole information obtained from a single NMR protocol. To analyze multicompartment exchanges, we propose a new strategy based on the simultaneous analysis of four inversion transfer protocols. The time course of ATP and Phosphocreatine (PCr) magnetizations computed from the McConnell equations were adjusted to their experimental value for exchange networks of increasing complexity (up to six metabolite pools). Exchange schemes were selected by the quality of their fit and their consistency with data from other sources: the size of mitochondrial pools and the ATP synthesis flux. The consideration of ATP-Pi exchange and of ATP compartmentation were insufficient to describe the data. The most appropriate exchange scheme in our normoxic heart involved the discrimination of three specific CK activities (cytosolic, mitochondrial, and close to ATPases). At the present level of heart contractility, the energy is transferred from mitochondria to myofibrils mainly by PCr.  相似文献   

5.
The exchange scheme of high energy phosphate transport in a whole heart relies on a system of CK functioning in different ways. This suggests that the CKs are able to act both like a shuttle and like a buffer for the energy transfer. The challenge is to understand how these two functions are balanced in the CK system. One key of this balance is the knowledge of the local concentrations of the ADP nucleotide. These concentrations cannot be directly measured, but they may be derived by computation. In the present report we introduce the known properties of the enzymes catalyzing the exchange of high energy phosphate into the model of flux pathways derived from NMR experiments to compute both the maximum activity of each enzyme and the local concentrations of all the substrates. We show that the ADP distribution must be heterogeneous for the system to work. Its concentration is 50% higher in the vicinity of ATPase sites and 50% lower in the intermembrane space of the mitochondria than in the cytosol. Another result of this analysis is that the apparent large unbalance of the CKmito pathway is imposed by the adenosine nucleotide transferase fluxes. This analysis proves that it is possible to deduce the local concentrations of a substrate by combining data originating from NMR, biochemistry and enzymology into a common model.  相似文献   

6.
A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle.Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM -/- showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils - this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result in the disturbances of the other component of the PCr pathway. In the latter case, energy is transferred from mitochondria to myofibrils by alternative metabolic pathways, probably involving adenylate kinase or other systems.  相似文献   

7.
In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstrated by an apparent discrepancy between the forward (F(f)) and reverse (F(r)) CK flux if this compartment was neglected in the analysis [Joubert et al. (2000) Biophys. J. 79, 1-13]. To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or gamma ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F(f)/F(r) ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP). Under conditions masking the influence of the ATP-P(i) exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.  相似文献   

8.
The mathematical model of the compartmentalized energy transfer system in cardiac myocytes presented includes mitochondrial synthesis of ATP by ATP synthase, phosphocreatine production in the coupled mitochondrial creatine kinase reaction, the myofibrillar and cytoplasmic creatine kinase reactions, ATP utilization by actomyosin ATPase during the contraction cycle, and diffusional exchange of metabolites between different compartments. The model was used to calculate the changes in metabolite profiles during the cardiac cycle, metabolite and energy fluxes in different cellular compartments at high workload (corresponding to the rate of oxygen consumption of 46 mu atoms of O.(g wet mass)-1.min-1) under varying conditions of restricted ADP diffusion across mitochondrial outer membrane and creatine kinase isoenzyme "switchoff." In the complete system, restricted diffusion of ADP across the outer mitochondrial membrane stabilizes phosphocreatine production in cardiac mitochondria and increases the role of the phosphocreatine shuttle in energy transport and respiration regulation. Selective inhibition of myoplasmic or mitochondrial creatine kinase (modeling the experiments with transgenic animals) results in "takeover" of their function by another, active creatine kinase isoenzyme. This mathematical modeling also shows that assumption of the creatine kinase equilibrium in the cell may only be a very rough approximation to the reality at increased workload. The mathematical model developed can be used as a basis for further quantitative analyses of energy fluxes in the cell and their regulation, particularly by adding modules for adenylate kinase, the glycolytic system, and other reactions of energy metabolism of the cell.  相似文献   

9.
The origin of the nuclear magnetic resonance (NMR)-measurable ATP in equilibrium Pi exchange and whether it can be used to determine net oxidative ATP synthesis rates in the intact myocardium were examined by detailed measurements of ATP in equilibrium Pi exchange rates in both directions as a function of the myocardial oxygen consumption rate (MVO2) in (1) glucose-perfused, isovolumic rat hearts with normal glycolytic activity and (2) pyruvate-perfused hearts where glycolytic activity was reduced or eliminated either by depletion of their endogenous glycogen or by use of the inhibitor iodoacetate. In glucose-perfused hearts, the Pi----ATP rate measured by the conventional two-site saturation transfer (CST) technique remained constant while MVO2 was increased approximately 2-fold. When the glycolytic activity was reduced, the Pi----ATP rate decreased significantly, demonstrating the existence of a significant glycolytic contribution. Upon elimination of the glycolytic component, the measured Pi----ATP rates displayed a linear dependence on MVO (micromoles of O consumption rate) with a slope of 2.36 +/- 0.15 (N = 8, standard error of the mean). This linear relationship is expected if the rate determined by CST is the net rate of ATP synthesis by the oxidative phosphorylation process, in which case the slope must equal the P:O ratio. The ATP----Pi rates and rate:MVO ratios measured by the multiple-site saturation transfer method at two MVO2 levels were equal to the corresponding Pi----ATP rates and rate:MVO ratios obtained in the absence of a glycolytic contribution. The following conclusions are drawn from these studies: (1) unless the glycolytic contribution to the ATP in equilibrium Pi exchange is inhibited or is specifically shown not to exist, the myocardial Pi in equilibrium ATP exchange due to oxidative phosphorylation cannot be studied by NMR; (2) at moderate MVO2 levels, the reaction catalyzed by the two glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase is near equilibrium; (3) the ATP synthesis by the mitochondrial H+-ATPase occurs unidirectionally (i.e., the reaction is far out of equilibrium); (4) the "operative" P:O ratio in the intact myocardium under our conditions is significantly less than the canonically accepted value of 3.  相似文献   

10.
Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by 18O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by 18O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K+ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2) ADP.  相似文献   

11.
ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with 31P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F0F1-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The 31P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to Pi by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of Pi was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular Pi to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the Pi signal as an intracellular pH-marker in plant cells.Abbreviations 3-PGA 3-phosphoglycerate - CW continuous wave - dG6P 2-deoxyglucose-6-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - MO equilibrium z-magnetization - M0 instantaneous z-magnetization after selective saturation for time t - MDP methylene-diphosphonic acid - PDE phosphodiester - PGK phosphoglycerate kinase - Pi inorganic orthophosphate - polyP polyphosphate - T1 longitudinal relaxation time - 1 longitudinal relaxation time with chemical exchange - TCA cycle tricarboxylic acid cycle Correspondence to: A. Mayer  相似文献   

12.
A network model for the determination of tumor metabolic fluxes from 13C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-13C2]glucose under normoxic conditions at 37 °C and monitored by 13C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.  相似文献   

13.
14.
It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by31 P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.  相似文献   

15.
Particulate fractions of Thiobacillus denitrificans catalyse the phosphorylation of ADP to ATP during the oxidation of various inorganic sulphur compounds or NADH via an electron transport chain. On the other hand, a soluble cell-free fraction synthesized ATP from APS and inorganic phosphate.The production of ATP was verified either by the firefly luciferin-luciferase enzyme system or by the incorporation of 32Pi into ATP. During the oxidation of sulphide, sulphite and NADH the production of ATP from ADP by particulate fractions is inhibited by compounds that inhibit electron transfer and by uncouplers of oxidative phosphorylation. However, these compounds had little effect on the production of ATP from AMP during the oxidation of sulphite by the soluble fraction. NADH was the most effective electron donor for oxidative phosphorylation. The soluble fraction contained high activities of ATP sulphurylase, inorganic pyrophosphatase and adenylate kinase but ADP sulphurylase activity was relatively low. The effects of inhibitors on ATP production from APS and Pi are compared with those on adenylate kinase and ATP sulphurylase.Abbreviations APS adenosine-5-phosphosulphate - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide  相似文献   

16.
Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state. The covalent bond forming and breaking step at the catalytic site (ADP + Pi in equilibrium ATP + HOH) appears relatively insensitive to uncouplers. Thus to the extent that uncouplers dissipate transmembrane proton-motive force, it is unlikely that such a force is used to drive ATP formation by direct protonations of Pi oxygens. When only Pi and ADP are added and formation of ATP from added ADP by adenylate kinase and subsequent ATP hydrolysis are adequately blocked, no Pi in equilibrium HOH exchange can be observed, demonstrating a requirement of energization by ATP binding and cleavage for such an exchange. This uncoupler-insensitive energization is suggested to represent a conformationally energized state that can be used reversibly to develop a transmembrane protonmotive force accompanying ADP and Pi release. Rates of various exchanges as estimated by improved procedures are compatible with all oxygen exchanges occurring by dynamic reversal of ATP hydrolysis at the catalytic site.  相似文献   

17.
The interpretation of creatine kinase (CK) flux measured by (31)P NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P(i) exchange on the forward (F(f): PCr --> ATP) and reverse (F(r)) CK fluxes derived from complete analysis of inversion transfer. Although F(f) should equal F(r) because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P(i) was masked by saturation of P(i) (sat-P(i)), F(f)/F(r) significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F(f)/F(r) for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P(i) might not result only from the masking of ATP-P(i) exchange.  相似文献   

18.
R Bowater  J Sleep 《Biochemistry》1988,27(14):5314-5323
The rate of ATP in equilibrium with Pi exchange, that is, the incorporation of medium Pi into ATP during the net hydrolysis of ATP, has been measured for rabbit psoas muscle fibers, myofibrils, and actomyosin subfragment 1 (acto-S1). The maximum exchange rate in fibers at saturating [Pi] is 0.04 s-1 per myosin head at 8 degrees C, pH 7, and an ionic strength of 0.2 M. The dependence of the rate on Pi concentration can be approximated by a hyperbola with an apparent dissociation constant (Km) of 3 mM. Myofibrils catalyze ATP in equilibrium with Pi exchange with a similar Km but at a slightly lower rate. In contrast, the soluble acto-S1 system, in which ATP hydrolysis is not coupled to tension generation, catalyzes exchange at a rate 500 times lower than that of fibers at low Pi concentration, and the Km for Pi is greater than 50 mM. The difference between the ATP in equilibrium with Pi exchange of fibers and of acto-S1 is discussed in terms of a model in which Pi binds to a force-generating state AM'-ADP and, due to mechanical constraint, the average free energy of this state is higher in the fiber than in acto-S1.  相似文献   

19.
Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.  相似文献   

20.
The role of the hydrolysis products of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and inorganic phosphate (Pi), in the control of myocardial respiration was evaluated in vivo using 31P NMR. These studies were conducted to evaluate whether increases in the ATP hydrolysis products can be detected through the cardiac cycle or during increases in cardiac work. 31P NMR data acquisitions gated to various portions of the cardiac cycle (50 msec time resolution) revealed that cytosolic ATP, ADP and Pi did not change over the course of the cardiac cycle. These metabolites were also monitored during steady-state increases in cardiac work in conjunction with measurements of coronary blood flow and oxygen consumption. No changes were observed during 2 to 3 fold increases in myocardial oxygen consumption induced by various methods. These results demonstrate that the cytosolic ATP, ADP, and Pi concentrations remain relatively constant throughout the cardiac cycle and during physiological increases in cardiac work and oxygen consumption. Furthermore, it is shown that ADP and Pi cannot be solely responsible for the regulation of cardiac respiration in vivo based on the in vitro Km values of these compounds for oxidative phosphorylation. It is concluded that other mechanisms, working in concert with the simple kinetic feedback of ATP hydrolysis products, must be present in the cytosol to provide control of myocardial respiration in vivo.  相似文献   

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