ATP-phosphocreatine metabolism catalyzed by creatine kinase. Comparison of saturation transfer (NMR) and isotope labeling technics

Unidirectional fluxes from ATP to phosphocreatine (PCr) catalyzed by MM-isoenzyme of creatine kinase (CK) were measured by using 31P-NMR saturation transfer technique and by means of radioactively labeled [gamma-32P]ATP. At 30-37 degrees C and pH 7.4 in a wide range of [PCr]/[creatine] ([PCr]/[Cr]) ratios (0.2 to 3.0) both of these methods gave similar results, thus showing that magnetization (saturation) transfer allows to determine fluxes close to real ones under "physiological" conditions. However, at [PCr]/[Cr] ratio higher than 5 ([ADP] less than 30 microM) or at decreased temperatures (7-15 degrees C, [PCr]/[Cr] approximately 1) fluxes determined by saturation transfer substantially exceeded those measured with the radioactive label. These data imply that under "physiological" conditions phosphoryl group transfer is actually rate-determining step of the CK reaction. On the contrary, at high [PCr]/[Cr] values or at low temperature the control step could be shifted from the phosphoryl group transfer or distributed among other steps of the reaction.     

Combination of 31P-NMR magnetization transfer and radioisotope exchange methods for assessment of an enzyme reaction mechanism: rate-determining steps of the creatine kinase reaction

The theoretical analysis of a reversible enzyme reaction performed in this work shows that the 31P-NMR magnetization (saturation) transfer technique combined with a radioisotope exchange method may potentially provide information on the position of rate-determining step(s). It depends on chemical shifts of NMR signals of nuclei of interest in free and enzyme-bound forms of substrate(s) and product(s) of the reaction. The creatine kinase reaction (MgATP + creatine----MgADP + P-creatine) has been used as a model. Chemical shifts of 31P in binary, ternary and transitional state substrate-enzyme complexes have been estimated by the variable frequency saturation transfer (VFST) method. This method is based on selective irradiation of numerous points in the spectrum and observation of changes in the intensity of visible line(s) which occur due to chemical exchange between it and lines which are not visible in the routine spectrum. Also, dissociation rate constants of MgADP-containing complexes were determined. Magnetization exchange rates, P-creatine----[gamma-P]MgATP and [beta-P]MgADP----[beta-P]MgATP, were compared with radioisotope exchange rates, [gamma-32P-MgATP----P-creatine and [3H]MgADP----MgATP at different [P-creatine]/[creatine] ratios and at different temperatures. All these exchange rates were close to each other at 30-37 degrees C and [PCr]/[Cr] ratios lower than 2. It is concluded that phosphoryl group transfer is the rate-determining step of the overall creatine kinase reaction under these conditions. However, at lower temperatures (below 25 degrees C) or at high [PCr]/[Cr] ratios ([ADP] less than 20 microM) the rate-determining step seems to be shifted toward dissociation of nucleotide substrates from enzyme-substrate complexes, since exchange rates became significantly different. This approach is useful for analysis of mechanism of enzymatic reactions and also can be applied to non-enzymatic reactions and evaluation of small rapidly exchangeable metabolite pools.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

The alpha beta-methylene analogues of ADP and ATP act as substrates for creatine kinase. delta G0 for this reaction and for the hydrolysis of the alpha beta-methylene analogue of ATP

The alpha beta-methylene analogues of ATP and ADP, [alpha beta CH2]ATP and [alpha beta CH2]ADP, are substrates for creatine kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10(-5)-times lower than that with normal ATP. The affinities of the analogues (especially [alpha beta CH2]ADP) for the enzyme are lower than those of the normal substrates. The equilibrium constant at 25 degrees C, measured using 31P NMR, for the reaction Mg[alpha beta CH2]ATP + creatine in equilibrium Mg[alpha beta CH2]ADP + phosphocreatine + H+ is 2.2 X 10(-12) M compared with a value of 2.5 X 10(-10) M for the same reaction with the normal substrates, corresponding to a difference in delta G0 values of 11.7 kJ X mol-1. It follows that delta G0 for the hydrolysis of the terminal phosphate group of Mg[alpha beta CH2]ATP is less favourable by 11.7 kJ X mol-1 than that for MgATP.     

Mathematical model of compartmentalized energy transfer: Its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice

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.     

Skeletal muscle metabolism and work capacity: a 31P-NMR study of Andean natives and lowlanders

Two metabolic features of altitude-adapted humans are the maximal O2 consumption (VO2max) paradox (higher work rates following acclimatization without increases in VO2max) and the lactate paradox (progressive reductions in muscle and blood lactate with exercise at increasing altitude). To assess underlying mechanisms, we studied six Andean Quechua Indians in La Raya, Peru (4,200 m) and at low altitude (less than 700 m) immediately upon arrival in Canada. The experimental strategy compared whole-body performance tests and single (calf) muscle work capacities in the Andeans with those in groups of sedentary, power-trained, and endurance-trained lowlanders. We used 31P nuclear magnetic resonance spectroscopy to monitor noninvasively changes in concentrations of phosphocreatine [( PCr]), [Pi], [ATP], [PCr]/[PCr] + creatine ([Cr]), [Pi]/[PCr] + [Cr], and pH in the gastrocnemius muscle of subjects exercising to fatigue. Our results indicate that the Andeans 1) are phenotypically unique with respect to measures of anaerobic and aerobic work capacity, 2) despite significantly lower anaerobic capacities, are capable of calf muscle work rates equal to those of highly trained power- and endurance-trained athletes, and 3) compared with endurance-trained athletes with significantly higher VO2max values and power-trained athletes with similar VO2max values, display, respectively, similar and reduced perturbation of all parameters related to the phosphorylation potential and to measurements of [Pi], [PCr], [ATP], and muscle pH derivable from nuclear magnetic resonance. Because the lactate paradox may be explained on the basis of tighter ATP demand-supplying coupling, we postulate that a similar mechanism may explain 1) the high calf muscle work capacities in the Andeans relative to measures of whole-body work capacity, 2) the VO2max paradox, and 3) anecdotal reports of exceptional work capacities in indigenous altitude natives.     

The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance.

1. The activity of creatine kinase in intact anaerobic frog muscle at 4 degrees C at rest and during contraction was investigated by using saturation-transfer 31P n.m.r. 2. At rest, the measured forward (phosphocreatine to ATP) reaction flux was 1.7 X 10(-3) M . s-1 and the backward flux was 1.2 X 10(-3) M . s-1. The large magnitude of both fluxes shows that creatine kinase is active in resting muscle, so the observed constancy of [phosphocreatine] demonstrates that the enzyme and its substrates are at equilibrium. 3. The apparent discrepancy between the fluxes must arise largely from an underestimation of the backward flux resulting from interaction of ATP with other systems, e.g. via adenylate kinase. For purposes of further calculation we have therefore adopted 1.6 X 10(-3) M . s-1 as an estimate of both fluxes. 4. During contraction, when the creatine kinase reaction is no longer at equilibrium, the net rate of phosphocreatine breakdown, estimated directly from the change in area of the inorganic phosphate peak, was 0.75 X 10(-3) M . s-1. Saturation transfer indicates that the forward reaction flux remains at approx. 1.6 X 10(-3) M . s-1 and the backward flux decreases to about 0.85 X 10(-3) M . s-1. 5. The activity of creatine kinase during contraction is large enough to account for the well-established observation that, during contraction, the concentration of ATP falls by less than 2-3%. The reaction catalysed by creatine kinase is driven forward during contraction by the large relative increase in the concentration of free ADP, which is more than doubled. 6. The observation that the forward flux does not increase during contraction and that the backward flux decreases can most simply be explained on the basis of competition of reactants for a limited amount of enzyme.     

Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.

We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.     

31P-NMR spectrum of phosphocreatine: Deuterium-induced splitting of the signal

It has been found in experiments with high resolution ³¹P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H₂O and D₂O and is single but with different chemical shifts in pure H₂O and D₂O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase.     

31P NMR saturation transfer measurements of phosphorus exchange reactions in rat heart and kidney in situ

31P NMR spectra of rat kidney and heart, in situ, were obtained at 97.2 MHz by using chronically implanted radio-frequency coils. Previous investigators have used magnetization transfer techniques to study phosphorus exchange in perfused kidney and heart. In the current experiments, saturation transfer techniques were used to measure the steady-state rate of exchange between inorganic phosphate (Pi) and the gamma-phosphate of ATP (gamma ATP) in kidney, and between phosphocreatine (PCr) and gamma ATP, catalyzed by creatine kinase, in heart. The rate constant for the exchange detected between Pi and gamma ATP in kidney, presumably catalyzed by oxidative phosphorylation, was 0.12 +/- 0.03 s-1. This corresponds to an ATP synthesis rate of 12 mumol min-1 (g wet weight)-1. Comparison of previously published O2 consumption and Na+ reabsorption rates for the intact kidney with the NMR-derived rate for ATP synthesis gave flux ratios of JATP/JO2 = 1.6-3.3 and JNa+/JATP = 4-10. The rate constants for the creatine kinase reaction, assuming a simple two-site exchange, were found to be 0.57 +/- 0.12 s-1 for the forward direction (PCr----ATP) and 0.50 +/- 0.16 s-1 for the reverse direction (ATP----PCr). The forward rate (0.78 +/- 0.18 intensity unit/s) was significantly larger (p less than 0.05) than the reverse rate (0.50 +/- 0.16 intensity unit/s). This difference between the forward and reverse rates of creatine kinase has been previously noted in the perfused heart. The difference has been attributed to participation of ATP in other reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Effects of 3-Nitropropionic Acid on Synaptosomal Energy and Transmitter Metabolism: Relevance to Neurodegenerative Brain Diseases

Abstract: 3-Nitropropionic acid (3-NPA) inhibited synaptosomal respiration in a dose-dependent manner; the degree of inhibition by the same concentration of the compound was greater, however, when respiration was stimulated by concomitant increase in ATP usage. The most rapid event after addition of 3-NPA was a decrease in [creatine phosphate]/[creatine] ([CrP]/[Cr]) and an increase in [lactate]/[pyruvate]. A fall in [ATP]/[ADP] and [GTP]/[GDP] was initially less pronounced but closely followed that in [CrP]/[Cr]. In the absence of glutamine, 3-NPA caused a pronounced decrease in internal aspartate level and a small reduction in glutamate concentration, whereas [GABA] rose; the sum of these three amino acids inside synaptosomes fell, but there were no increases in their external levels. With glutamine in the medium, the reduction in intrasynaptosomal aspartate was accompanied by increases in intrasynaptosomal glutamate and GABA. The external concentration of glutamate rose substantially in the presence of the inhibitor. 3-NPA had no effect on basal release of either glutamate (and GABA) or biogenic amines but increased efflux occurring upon addition of nonsaturating concentrations of the depolarizing agents veratridine and KCI. The results allow the following predictions with respect to the behavior of brain metabolism in neurodegenerative diseases that involve restrictions of mitochondrial function: (1) The extent of inhibition of mitochondrial ATP generation is expected to be greater in cells with high energy demand. The earliest signs of impairment of the respiratory chain function are a fall in [PCr]/[Cr] (or a rise in [Pi]/[CrP]) and an increase in [lactate]/[pyruvate]. (2) A fall in [GTP]/[GDP] can limit protein synthesis. This may be one of the factors that contributes to cell death. (3) An increase in the concentration of inorganic phosphate stimulates neuronal glutaminase activity and leads to a release of glutamate into the external environment; the latter could activate excitatory amino acid receptors. (4) A lowered energy level limits the cell's ability to restore ion gradients. Stimulated release of transmitters from neurons may, therefore, be enhanced and their reuptake delayed.     

Kinetics of creatine kinase in heart: a 31P NMR saturation- and inversion-transfer study

The kinetics of the phosphate exchange by creatine kinase (CK) was studied in solution and in the Langendorff-perfused rat heart at 37 degrees C. 31P inversion-transfer (IT) and saturation-transfer (ST) methods were applied. The kinetic parameters obtained by the two magnetization transfer methods were the same, whether in solution or in the perfused heart. Inversion transfer is the more efficient method, yielding the kinetic constants for the exchange and the relaxation rates of the transferred phosphate in both substrates, in one experiment. In solution the forward (kF) and reverse (kR) pseudo-first-order rate constants for the CK reaction (kF = k1[MgADP][H+]; kR = k-1[creatine]) as well as the concentrations of phosphocreatine (PCr), MgATP, and creatine (Cr) remained constant between pH 6.9 and pH 7.8. Equilibrium at this pH region is therefore maintained by compensating changes in the concentration of MgADP. The forward and reverse fluxes in the perfused heart were equal with an average flux ratio (fluxF/fluxR) of 0.975 +/- 0.065 obtained by both methods. Average values of kF and kR were 0.725 +/- 0.077 and 1.12 +/- 0.14 s-1, respectively. These results clearly indicate that the CK reaction in the Langendorff-perfused heart is in equilibrium and its rate is not limited by the diffusion of substrates between different locations of the enzyme. There is therefore no indication of compartmentation of substrates of the CK reaction.     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

31P NMR studies of the arginine kinase reaction. Equilibrium constants and exchange rates at stoichiometric enzyme concentration.

The arginine kinase reaction, the reversible transfer of the terminal phosphoryl group of ATP to L-arginine, has been investigated by the technique of 31P NMR at catalytic and stoichiometric concentrations of the enzyme. Three of the four substrates, ATP, ADP, and P-arginine produce easily distinguishable resonances in the 31P NMR spectrum, thus permitting a determination of equilibrium constants from the integrated areas of the resonances. From the linewidths, the exchange rates between reactants and products may be evaluated. At pH 7.25 and a temperature of 12 degrees, the equilibrium constant at catalytic enzyme concentration: Keq = [MgADP] [P-arginine]/[MgATP] [L-arginine], is found to be 0.10 +/- 0.02 and that at stoichiometric enzyme concentration: K'eq = [E-MgADP] [E-P-arginine]/[E-MgATP] [E-arginine] to be 1.56 +/- 0.5. Thus, as the enzyme concentration increased, the production of P-arginine is increasingly favored. From the NMR line shapes in the presence of excess enzyme, the rate of the single step, the transfer of the phosphoryl group on the surface of the enzyme is found to be 192 +/- 15 s-1 in the forward direction, i.e. from E-MgATP, and 154 +/- 15 s-1 in the reverse direction from E-P-argine. At 12 degrees and pH 7.25, the rate of the overall reaction in the forward direction was determined from kinetic measurements to be 19 s-1, an order of magnitude slower than the rate measured by NMR. It can, therefore, be concluded that the interconversion of substrates on the surface of the enzyme is not the rate-determining step in the overal reaction. From the equilibrium constants and other known data the dissociation constant of P-arginine from its enzyme complex can be determined and is found to be 100 muM.     

Systems bioenergetics of creatine kinase networks: physiological roles of creatine and phosphocreatine in regulation of cardiac cell function

Physiological role of creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of creatine and with this results in phosphocreatine (PCr) production with PCr/O₂ ratio of about 5–6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank–Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by mitochondrial creatine kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized creatine kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.     

31P saturation transfer spectroscopy predicts differential intracellular macromolecular association of ATP and ADP in skeletal muscle

The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo ³¹P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in ³¹P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of ³¹P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of β-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP ↔ ATP exchange, in which ADP is only transiently present in the cytosol.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.