共查询到20条相似文献,搜索用时 15 毫秒
1.
Pucar D Dzeja PP Bast P Gumina RJ Drahl C Lim L Juranic N Macura S Terzic A 《Molecular and cellular biochemistry》2004,256(1-2):281-289
Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress. 相似文献
2.
31P NMR spin-transfer in the phosphoglyceromutase reaction 总被引:3,自引:0,他引:3
The rate of exchange of phosphoryl groups between 2- and 3-phosphoglycerate catalysed by (a) high concentrations (approximately equal to 5.0 mg protein ml-1) of rabbit muscle phosphoglyceromutase and (b) lysed human erythrocytes was measured using saturation and inversion transfer techniques with 31P-NMR spectroscopy. This is the first reported application of these techniques to a study of this particular enzymic reaction either in vitro or in situ in a cell cytosol. Selective irradiation of resonances was achieved by the DANTE pulse sequence which had not previously been used for spin-transfer studies. New equilibrium exchange theory was developed for the simplest model of a two-reactant enzyme-catalysed reaction and this was used to calculate turnover rates for the enzymes. There was a close similarity between the turnover rates calculated from the spin-transfer data obtained from the systems in vitro and in situ and those obtained by conventional enzymic assays, at low enzyme concentrations. This suggested an absence of any homogeneous enzyme-enzyme interactions which modify the kinetics at high protein concentrations either in lysates or in the system in vitro. 相似文献
3.
Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. 31P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in 31P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid 31P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible 31P NMR spectra of phospholipids. 相似文献
4.
Stumber M Geyer M Graf R Kalbitzer HR Scheffzek K Haeberlen U 《Journal of molecular biology》2002,323(5):899-907
The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3mm(3) size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10(15) phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras-nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras-GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal. 相似文献
5.
31P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. Our results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (Pi) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP gamma saturation and were used in combination with ST data to determine Pi consumption rates. 13C NMR and O2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O2 consumption, respectively, under the various metabolic conditions examined. Our results suggest that GAPDH/PGK-catalyzed Pi-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally. 相似文献
6.
Higgins L Korzekwa KR Rao S Shou M Jones JP 《Archives of biochemistry and biophysics》2001,385(1):220-230
Regioselectivity is used to determine the absolute energetic differences for four different reactions catalyzed by P450. Abstraction of a hydrogen from a benzylic carbon containing a chlorine has a 1.0 kcal/mol lower barrier than abstraction from a simple benzylic carbon, which in turn is 0.4 to 0.9 kcal/mol lower than abstraction from the methyl group of an aromatic ether and 0.1 to 0.6 kcal/mol easier than aromatic hydroxylation. Isotope effects are used to determine if the enzyme-substrate complexes leading to each product, from a given substrate, are in rapid equilibrium. For all enzymes isotopically sensitive branching is observed from the benzylic carbon upon deuterium incorporation at that position to each of the other positions, indicating that each product arises from the same active oxygen species. The energetic differences determined experimentally are accurately reproduced by theoretical hydrogen atom abstractions at both the AM1 semiempirical and DFT levels of theory. 相似文献
7.
SIp NMR studies on microorganisms have been carried out with the cells embedded in agarose gel. The novel use of the gel for the NMR studies has advantages over the usual liquid suspensions in terms of improved reproducibility of data and cell viability, with no net loss of spectral quality. Polyphosphate formation in Escherichia coli was monitored continuously for up to 24 h and metabolic changes in yeast for 6 h. Changes of the intracellular pH during glycolysis in yeast were determined from the chemical shift of the internal Pi. NMR titration curves of Pi in the presence of Mg2+ indicate uncertainties in internal pH values estimated by this technique. 相似文献
8.
9.
Gerhard Wegener Nicholas M. Bolas André A. G. Thomas 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(3):247-256
Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of 31P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.
Dedicated to Professor Dr. Ernst Zebe (University of Münster) on occasion of his 65th birthday. 相似文献
1. | In the flight muscle of resting locusts the ratio of phosphoarginine to ATP was the same whether determined by NMR (1.76) or biochemically, but the NMR-visible content of inorganic phosphate (Pi) was only 40% of ATP, i.e., much lower than total Pi as determined biochemically. This suggests that most of the Pi in flight muscle is not free, and hence not available as substrate or effector for cytosolic enzymes. Similarly, the free content of ADP and AMP in resting muscle was calculated to be much lower than the total content. |
2. | Flight brought about a marked increase in Pi and a decrease in phosphoarginine in flight muscle although there was no change in intracellular pH. |
3. | At the initiation of flight a new steady state of ATP, Pi, and phosphoarginine was rapidly established and minimal changes occurred after the first 2 s of flight. |
4. | From the free contents of ATP and phosphoarginine in working flight muscle the flight-induced fractional increases in free ADP and free AMP were calculated to be 5.0-fold and 27.4-fold, respectively. As Pi, ADP, and AMP are substrates and potent effectors of enzymes, the flight-induced increase in their contents is likely to have marked effects on metabolic flux in working muscle. |
5. | After short-term flight as well as prolonged flight, phosphoarginine, ATP, and Pi returned rapidly to their preflight levels, indicating that metabolic recovery from flight is rapid. |
6. | The locust appears to be an appropriate model for the study of metabolic regulation in aerobic muscle during exercise. |
10.
P F Daly R C Lyon P J Faustino J S Cohen 《The Journal of biological chemistry》1987,262(31):14875-14878
Addition of choline, ethanolamine, or hemicholinium-3 (a choline kinase inhibitor) to the perfusate of human breast cancer cells monitored by 31P NMR spectroscopy resulted in significant changes to phosphomonoester (PME) and phosphodiester (PDE) signals. These results enable us to assign the PMEs to phosphcholine (PC) and phosphoethanolamine (PE), the PDEs to glycerophosphorylcholine and glycerophosphorylethanolamine, and to define the pathways producing them. The PMEs are products of choline and ethanolamine kinases, the first steps in phospholipid synthesis; and the PDEs are substrates of glycerophosphorylcholine phosphodiesterase, the last step in phospholipid catabolism. Furthermore, PC and PE peaks are twice as intense in cells at log phase versus confluency. We also observed these signals in vivo in human colon and breast tumors grown in mice. Since PMEs are low in most nonproliferating tissues, they could form a basis for noninvasive diagnosis. Also, PE and PC are situated between the control enzymes of two major synthetic pathways and will allow noninvasive 31P NMR studies of these pathways in intact cells and in vivo. 相似文献
11.
12.
Nanna Rasmussen David C. Lloyd R. George Ratcliffe Poul Erik Hansen Iver Jakobsen 《Plant and Soil》2000,226(2):245-253
31P nuclear magnetic resonance (NMR) spectroscopy was used to study phosphate (P) metabolism in mycorrhizal and nonmycorrhizal
roots of cucumber (Cucumis sativus L) and in external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. The in vivo NMR method allows biological systems to be studied non-invasively and non-destructively. 31P NMR experiments provide information about cytoplasmic and vacuolar pH, based on the pH-dependent chemical shifts of the
signals arising from the inorganic P (Pi) located in the two compartments. Similarly, the resonances arising from α, β and γ phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity
and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with
this method. The 31P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in
the spectra of nonmycorrhizal roots. This demonstrated that the Pi taken up by the fungus was transformed into PolyP with a short chain length. The spectra of excised AM fungi revealed only
a small signal from the cytoplasmic Pi, suggesting a low cytoplasmic volume in this AM fungus.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
13.
Kinetics of oxygen-18 exchange between inorganic phosphate and water catalyzed by myosin subfragment 1, using the 18O shift in 31P NMR 总被引:1,自引:0,他引:1
The time course of oxygen-18 exchange between [18O]Pi and normal water, catalyzed by myosin subfragment 1 in the presence of MgADP, was followed using the shift in 31P NMR caused by the presence of oxygen-18 bound to the phosphorus. Essentially all molecules of [18O]Pi that bind to the enzyme undergo complete exchange and are released as [16O4]Pi. Exchange probably occurs by formation of myosin.ATP from a myosin.ADP.Pi complex and is rapid relative to release of Pi from this complex. The kinetics of exchange give a value for the rate constant for binding Pi to myosin.ADP of 0.23 M-1 S-1 (pH 8.0, 22 degrees C). This value is consistent with exchange occurring by reversal of the ATP-ase reaction back to the myosin.ATP complex. 相似文献
14.
The intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT)-(L)-tryptophan methyl ester phosphoramidate (L-ATO) and AZT-(L)-phenylalanine methyl ester phosphoramidate (L-APO) by the human T-lymphoblastoid cell line CCRF-CEM (CEM-1.3) and peripheral blood mononuclear cell line (PBMC) was investigated with high field 31P NMR spectroscopy. The AZT amino acid phosphoramidates were shown to accumulate intracellularly and to be readily converted into AZT-MP by both tissues types. Thus, the efficient delivery of nucleoside monophosphates to cells can be facilitated by nucleoside phosphoramidate pronucleotides. 相似文献
15.
The role of escape reactions in determining the size distribution of prey captured by herring larvae
Michael R. Heath 《Environmental Biology of Fishes》1993,38(4):331-344
Synopsis Fish larvae are selective planktivores, and size is an important factor in prey selection. However, for herring larvae, a selection model based solely on the principle of optimising calorific gain per unit energy expenditure consistently overestimates the mean size of ingested prey. Most such models ignore the escape capabilities and shape of the prey, and the potential role of escape on capture success. In this paper, a static foraging model incorporating both selection and escape is described and tested against published data on the composition of herring larvae stomach contents. The results indicate that prey escape is a major factor structuring the diet composition of the larvae. 相似文献
16.
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) 相似文献
17.
The metabolic alterations induced, in the isolated rat heart, by graded ischaemia and reperfusion, were evaluated both by 31P-NMR spectroscopy and by biochemical analysis. The relative changes in phosphorylated compound contents measured by both methods were well correlated for ATP (r = 0.94) and Pi (r = 0.88), but less for PC (r = 0.72). These results demonstrate that the data drawn from the 2 methods compare well, for the assessment of the effects of ischaemia on the isolated heart. In order to characterize the extent or energetic alterations caused by ischaemia and reperfusion from the data drawn from a single NMR spectrum various indices have been calculated. A simple index (Formula: see text) seems to this regard, as discriminative as the adenylate charge or the phosphorylation potential. 相似文献
18.
Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected. 相似文献
19.
《生物化学与生物物理学报:生物膜》2015,1848(3):760-766
Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used 31P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and 31P T2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T2 relaxation are observed at higher temperatures. A comparison of 31P T1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules – in particular for the negatively charged DMPG – while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide–membrane interactions. 相似文献