Role of creatine phosphokinase in the energy supply of the pumping function of the heart]

The cardiac output of isolated working rat heart and left ventricular pressure were estimated in either almost complete inhibition of creatine kinase by iodoacetamide or predominant fall in adenine nucleotides (AdN) content induced by 2-deoxyglucose treatment. In the former case, a profound cardiac pump failure was observed despite almost normal levels of myocardial AdN and phosphocreatine. Those hearts could not maintain the aortic output at standard load due to lower LV systolic pressure, that was accompanied by increased minimal and maximal diastolic pressures by 5-7 mm Hg as well as by LV diastolic stiffness. As LV systolic pressure in those hearts was unchanged in retrogradely perfused and unloaded hearts it might be suggested that the cardiac pump failure was caused by the decreased LV distensibility. On the contrary, deoxyglucose treatment that resulted in 70% fall in the AdN content was accompanied by only moderate reduction of the cardiac output and insignificant changes in LV diastolic pressure and stiffness. The results suggested that creatine kinase plays a crucial role in the maintenance of normal myofibrillar compliance, which is necessary for cardiac filling and pump function.     

L-Pyroglutamic Acid Inhibits Energy Production and Lipid Synthesis in Cerebral Cortex of Young Rats In Vitro

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO₂ production and lipid synthesis from [U-¹⁴C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO₂ production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.     

AT1receptor blockade alters metabolic,functional and structural proteins after reperfused myocardial infarction: Detection using proteomics

Angiotensin II (AngII) type 1 receptor (AT₁R) blockers (ARBs) limit left ventricular (LV) dysfunction and necrosis after reperfused myocardial infarction (RMI) and proteomics can detect changes in protein levels after injury. We applied proteomics to detect changes in levels of specific protein in the ischemic zone (IZ) and non-ischemic zone (NIZ) of dog hearts after in vivo RMI (90 min of anterior ischemia; 120 min of reperfusion) and treatment with intravenous vehicle (control) and the ARBs valsartan or irbesartan (10 mg/kg) over 30 min before RMI. We also assessed LV function, infarction and apoptosis. Both ARBs limited the RMI-induced LV dysfunction, infarct size and apoptosis. Proteomics detected differential expression of 5 randomly selected proteins in the IZ compared to the NIZ after RMI: decrease in subunit of ATP synthase isoform precursor (consistent with increased conversion to subunit under metabolic stress), M chain creatine kinase (consistent with cellular damage) and ventricular myosin light chain-1 (consistent with structural damage and decreased contractility); and increase in NAD⁺-isocitrate dehydrogenase (ICDH) and subunit and ATP synthase D chain (mitochondrial, consistent with metabolic dysfunction). Importantly, changes in NAD₊-ICDH and ATP synthase D chain were reversed by ARB therapy. Thus, proteomics can detect regional changes in metabolic, contractile, and structural proteins after RMI and several of these proteins are favorably modified by ARBs, suggesting that they may be novel therapeutic targets. (Mol Cell Biochem 263: 179–188, 2004)     

Molecular Properties of Purified Human Uncoupling Protein 2 Refolded from Bacterial Inclusion Bodies

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (K _d) of 0.3–0.5 and 23–92 M. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a K _d of greater than 100 M, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-¹⁴C] ATP and [8-¹⁴C] ADP with K _ds of 4–5 and 16–18 M, respectively. Mg²⁺ (2 mM) reduced the apparent ATP affinity to 53 M, an effect entirely explained by chelation of ATP; with Mg²⁺, K _d using calculated free ATP was 3 M. A combination of gel filtration, Cu²⁺-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg²⁺-free nucleotides: the K _d for ATP is about 3–5 M and for Mant-ATP it is about 10 times lower; and (c) in C₁₂E₉ detergent, the monodisperse protein may be in dimeric form.     

Ribose Supplementation Alone or with Elevated Creatine Does Not Preserve High Energy Nucleotides or Cardiac Function in the Failing Mouse Heart

Background

Reduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP) are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE).

Methods and Results

Four groups were studied: sham; myocardial infarction (MI); MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN) pool was decreased to a similar amount (8–14%) in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV) dysfunction (3-fold reduction in ejection fraction) and LV hypertrophy (32–47% increased mass). Ejection fraction closely correlated with infarct size independently of treatment (r² = 0.63, p<0.0001), but did not correlate with myocardial creatine or TAN levels.

Conclusion

Elevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated.     

Regulation of ATP-stimulated releasable myofilaments from cardiac and skeletal muscle myofibrils

The mechanism underlying the formation of easily releasable myofilaments, from myofibrils treated with an ATP-containing relaxing solution, was examined in this investigation. The proportion of releasable myofilaments purified from myofibrils of cardiac, fast- and slow-twitch muscles increased as the [ATP] was raised from 0 to 8.5 mM. The protein composition of the easily releasable myofilaments did not differ with increasing ATP concentrations as observed by 5–15% linear gradient SDS-PAGE. There is a nucleotide specificity to the release of myofilaments in the order of ATP > GTP >> UTP > CTP. Experiments with AMP-PNP and inorganic phosphate (Pi) showed that ATP hydrolysis and the build up of Pi are not requirements in the formation of the easily releasable myofilaments. The release of myofilaments was found to be insensitive to variations in pH from 6.5 to 7.5. The ATP stimulation of myofilament release is ubiquitin-independent, since incubation of purified myofibrils with ubiquitin (1–100 g/ml) at both 20 and 37°C did not change the amount released. Modifying the free sulfhydryl group content by treatment of myofibrils with NEM (0.01–1 mM) or silver nitrate (0.1–10 mM) decreased the proportion of myofilaments that were releasable. Exclusion of 1 mM DTT from the preparation of myofibrils had similar results. These results indicate that the formation of easily releasable myofilaments can be mediated by metabolically related parameters such as the adenosine nucleotides and the reduction-oxidation status of the myofibrillar proteins of striated muscle.     

Macrocompartmentation of total creatine in cardiomyocytes revisited

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments – mitochondrial matrix space and cytoplasm – in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 g/ml for 30 min or 600 g/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2–5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 ± 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment – cytoplasm.     

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/ myofibril system in muscle

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional coupling between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters and that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible coupling are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when or is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when or is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the adenine nucleotide flux at the ends of the model into creatine flux in the middle; the overall net flux is small, but only zero if = . A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.     

Current-voltage relationships for the plasma membrane and its principal electrogenic pump inNeurospora crassa: I. Steady-state conditions

Summary The nonlinear membrane current-voltage relationship (I–V curve) for intact hyphae ofNeurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus quasi-linear cable theory. Under normal conditions of growth and respiration, the membraneI–V curve is best described as a parabolic segement convex in the direction of depolarizing current. At the average resting potential of –174 mV, the membrane conductance is 190 mhos/cm²; conductance increases to 240 mhos/cm² at –300 mV, and decreases to 130 mhos/cm² at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range.Inhibition of the primary electrogenic pump inNeurospora by ATP withdrawal (with 1mm KCN) depolarizes the membrane to the range of –40 to –70 mV and reduces the slope of theI–V curve by a fixed scaling factor of approximately 0.8. For wild-typeNeurospora, compared under control conditions and during steady-state inhibition by cyanide, theI–V difference curve — presumed to define the current-voltage curve for the electrogenic pump — is a saturation function with maximal current of 20 A/cm², a half-saturation potential near –300 mV, and a projected reversal potential of ca. –400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H⁺ extruded:1 ATP split.The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-stateI–V curves, under the assumption that cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects.The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 F/cm²) a second of 50–75 msec; and a third of 20–30 sec, perhaps representing changes of intracellular composition.     

Identification of subcellular energy fluxes by P NMR spectroscopy in the perfused heart: contractility induced modifications of energy transfer pathways

The identification of subcellular fluxes of exchange of ATP, phosphocreatine (PCr) and Pi between mitochondria, cytosol and ATPases and pathways of energy transfer in a whole organ is a challenge specially in the myocardium where 50% of creatine kinases (CK) are found in close vicinity of ATP producing (mito-CK) and utilizing ( MM-bound CK) reactions. To dissect their contribution in cardiac energy transfer we recently developed a new experimental³¹P NMR spectroscopy approach. This led to identify three kinetically different subcellular CKs and to evidence experimentally the CK shuttle in a rat heart perfused in isovolumy. Here we show that a decreased energy demand alters energetic pathways : two CKs (cytosolic and MM-bound) functioning at equilibrium and a non mitochondrial ATPPi exchange was sufficient to describe NMR data. Mito-CK fluxes was not detected anymore. This confirms the dependence of energy pathways upon cardiac activity. Indeed the subcellular localization and activity of CKs may have important bioenergetic consequences for the in vivo control of respiration at high work: free ADP estimated from global CK equilibrium might not always adequately reflect its concentration at the ANT.     

A proton-translocating adenosine triphosphatase is associated with the peroxisomal membrane of yeasts

The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical analysis revealed that ATPase activity was associated with the peroxisomal peak fractions which were identified on the basis of alcohol oxidase and catalase activity. The properties of this ATPase closely resembled those of the mitochondrial ATPase of this yeast. The enzyme was Mg²⁺-dependent, had a pH optimum of approximately 8.5 and was sensitive to N,N-dicyclohexylcarbodiimide (DCCD), oligomycin and azide, but not to vanadate. A major difference was the apparent K _m for ATP which was 4–6 mM for the peroxisomal ATPase compared to 0.6–0.9 mM for the mitochondrial enzyme.Cytochemical experiments indicated that the peroxisomal ATPase was associated with the membranes surrounding these organelles. After incubations with CeCl₃ and ATP specific reaction products were localized on the peroxisomal membrane, both when unfixed isolated peroxisomes or formaldehyde-fixed protoplasts were used. This staining was strictly ATP-dependent; in controls performed i) in the absence of substrate, ii) in the presence of glycerol 2-phosphate instead of ATP, or iii) in the presence of DCCD, staining was invariably absent. Similar staining patterns were observed in subcellular fractions and protoplasts of Candida utilis and Trichosporon cutaneum X4, grown in the presence of ethanol/ethylamine or ethylamine, respectively.Abbreviations MES 2-(N-Morpholino)ethanesulfonic acid - DCCD N,N-dicyclohexylcarbodiimide     

Cardiac phosphocreatine deficiency induced by GPA during postnatal development in rat

The effect of chronic administration of -guanidinopropionic acid (GPA) on the protein profiling, energy metabolism and right ventricular (RV) function was studied in the rat heart during the weaning and adolescence period. GPA was given in tap water (1–1.5%) using pair drink controls. The feeding of animals with GPA solution for a six week period resulted in elevation of heart to body weight ratio due to body growth retardation. GPA accumulated in the myocardium up to 67.37 ± 5.3 moles.g dry weight and the tissue content of total creatine, phosphocreatine and ATP was significantly decreased to 15%, 9% and 65% of control values respectively. Total activity of creatine kinase (CK) was not changed, but the proportion of mitochondrial (Mi) CK isoenzyme was decreased; the percentage of MB isoenzyme of CK was significantly higher. GPA treatment resulted in an elevation of the content of cardiac collagenous proteins and decrease of non-collagenous proteins in the heart; in parallel, a decrease of the collagen I to collagen III ratio was detected. The function of the RV was assessed using an isolated perfused heart with RV performing pressure-volume work. As compared to pair-drink controls, RV function was significantly impaired the GPA group: at any given right atrial filling pressure, the RV systolic pressure and the rate of pressure development were decreased by almost a factor of two. Elevation of the RV diastolic pressure with increasing pulmonary artery diastolic pressure was also significantly steeper in the GPA group which also showed decrease of cardiac output, especially at high outflow resistance. It may be assumed that chronic administration of GPA deeply influenced metabolic parameters, protein profiles and contractile function of the developing heart. On the other hand, concentrations of glucose, total lipids and triglycerides in blood plasma were not affected. All these data confirm the concept that the CK system is of central importance both for heart function and for the regulation of normal growth of cardiac myocytes.     

The effect of exogenous N6-(Δ2-isopentenyl)adenine on aerobic energy generation in Zymomonas mobilis

A direct linear relationship between the rate of oxygen consumption and ATP content in starved Zymomonas mobilis cells was observed in the presence of ethanol (0.056–1.12 mM) as the substrate. Both the rate of oxygen consumption and the ATP content were significantly reduced by the exogenously added plant growth substance N⁶-(²-isopentenyl)adenine (i⁶Ade), directly proportional to the concentration (0.125–0.5 mM) of i⁶Ade in the incubation medium. The results obtained are consistent with the current view of ATP synthesis by oxidative phosphorylation in non-growing Z. mobilis cells and gives evidence that i⁶Ade can be used as a tool to affect in vivo the alcohol dehydrogenase reaction, which provides reducing equivalents for ethanol-dependent aerobic energy generation.     

Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock

Effects of endotoxin administration on the ATP-dependent Ca²⁺ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca²⁺ uptake by cardiac SR was decreased by 27–43% (p < 0.05). Kinetic analysis indicates that the Vmax values for Ca²⁺ and for ATP were significantly decreased while the S_0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1–5 mM) stimulated while vanadate (25–50 M) inhibited the ATP-dependent Ca²⁺ uptake, but the Mg²⁺-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca²⁺ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca²⁺ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p < 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca²⁺ uptake in canine cardiac SR and the endotoxininduced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca²⁺ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

ATP,creatine phosphate,and mechanical activity in rainbow trout myocardium under inhibition of glycolysis and cell respiration

Summary Twitch force and resting tension of electrically stimulated ventricular strips of rainbow trout were compared with tissue contents of phosphocreatine, creatine, and ATP. The phosphocreatine/total creatine ratio, which was used to assess the cytoplasmic phosphorylation potential, fell with the fraction of cell respiration that was inhibited by sodium cyanide and N₂. Concomitantly, twitch force decreased while resting tension tended to increase. This relation between phosphocreatine/total creatine and mechanical parameters became more prominent as glycolysis was increasingly inhibited by sodium iodoacetate. Furthermore, glycolytic inhibition was followed by a decrease in the ATP/phosphocreatine ratio. The latter effect was the same in 1% and 6% CO₂. Thus, it cannot be ascribed to an action of intracellular pH on the creatine kinase catalyzed reaction. Notably, resting tension as well as twitch force relative to ATP was augmented by glycolytic inhibition. The main conclusions are that in the presence of a decreased mitochondrial activity, glycolysis protects contractility not only by counteracting a lowering in high energy phosphates but also by supporting the ATP/phosphocreatine ratio. Apparently, the creatine kinase activity is insufficient to maintain ATP in equilibrium with phosphocreatine. In addition, glycolysis seems to elevate the level of free phosphate relative to ATP, so that twitch force development as well as rigor complex formation is counteracted.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Expressing creatine kinase in transgenic tobacco – a first step towards introducing an energy buffering system in plants

Creatine kinase a key enzyme in cellular energy homeostasis of vertebrates offers the promise of engineering plants with enhanced stress tolerance. In order to provide plants with such an energy buffering system, tobacco was transformed with a cDNA, encoding the cytosolic brain-type isoform of chicken creatine kinase (BB-CK), the expression of which was under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Transgenic tobacco plants were selected and suspension cultures generated. Both transgenic plants and suspension cultures were shown to stably express enzymatically active BB-CK in vitro and in vivo, and in most cases for three successive generations (T₀–T₂). Exogenously supplied creatine was shown to enter the plant cells and resulted in only a slight reduction in root growth at concentrations up to 10mM. Furthermore, the BB-CK expressing tobacco plants and cell suspension cultures were able to convert creatine into phosphocreatine.     

Role of energy in oxidative phosphorylation

This article reviews the current status of information regarding the role of energy in the process of oxidative phosphorylation by mitochondria. The available data suggest that in submitochondrial particles (SMP) energy is utilized for the binding of ADP and P_i and for the release of ATP bound at the catalytic sites of F₁-ATPase. The process of ATP synthesis on the surface of F₁ from F₁-bound ADP and P_i appears to be associated with negligible free energy change. The rate of energy production by the respiratory chain modulates the kinetics of ATP synthesis between a lowK _m (for ADP and P_i)-lowV _max mode and a highK _m -highV _max mode. TheK _m extremes for ADP are 2–3 µM and 120–150 µM, andV _max for ATP synthesis at high rates of energy production by bovine-heart SMP is about 440 s^–1 (mole F₁)^–1 at 30°C, which corresponds to 11 µmol ATP (min · mg of protein)^–1. The interaction of dicyclohexylcarbodiimide (DCCD) or oligomycin at the proteolipid (subunitc) of the membrane sector (F₀) of the ATP synthase complex alters the mode of ATP binding at the catalytic sites of F₁, probably to one of lower affinity. It has been suggested that protonic energy might be conveyed to the catalytic sites of F₁ in an analogous manner, i.e., via conformation changes in the ATP synthase complex initiated by proton-induced alterations in the structure of the DCCD-binding proteolipid. Finally, the relationship between the steady-state membrane potential () and the rates of electron transfer and ATP synthesis has been discussed. It has been shown, in agreement with the delocalized chemiosmotic mechanism, that under appropriate conditions is exquisitely sensitive to changes in the rates of energy production and consumption.