Exercise-induced splitting of the inorganic phosphate peak: investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy

To investigate the splitting of the inorganic phosphate (Pi) peak during exercise and recovery, a time-resolved ³¹phosphorus nuclear magnetic resonance spectroscopy (³¹P-MRS) technique was used. Seven healthy young sedentary male subjects performed knee flexion exercise in the prone position inside a 2.1-T magnet, with the surface coil for ³¹P-MRS being placed on the biceps femoris muscle. After a 1-min warm-up without loading, the exercise intensity was increased by 0.41 W at 15-s intervals until exhaustion, followed by a 5-min recovery period. The ³¹P-MRS were recorded every 5 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of the Pi and phosphocreatine peaks were performed. Five of the seven subjects showed two distinct Pi peaks during exercise, suggesting two different pH distributions in exercising muscle (high pH and low pH region). In these five subjects, the high-pH increased rapidly just after the onset of exercise, while the low-pH peak increased gradually approximately 60 s after the onset of exercise. During recovery, the disappearance of the high-pH peak was more rapid than that of the low-pH peak. These findings suggest that our method ³¹P-MRS provides a simple approach for studying the kinetics of the Pi peak and intramuscular pH during exercise and recovery.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these 30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.     

Effects of L-carnitine and its acetyl and propionyl esters on ATP and PCr levels of isolated rat hearts perfused without fatty acids and investigated by means of 31P-NMR spectroscopy

³¹P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the P_i signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/P_i ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/P_i ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/P_i ratio was observed. Intracellular pH can be calculated from determination of the P_i-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases.     

Responses of mouse fast and slow skeletal muscle to streptozotocin diabetes: Myosin isoenzymes and phosphorous metabolites

A condition similar to insulin-dependent diabetes mellitus (IDDM) was induced in male CD-1 mice by injection of streptozotocin (STZ). Five weeks after treatment, the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles were isolated for analysis. Phosphorous metabolites were quantified by³¹P-NMR and HPLC, native myosin was characterized electrophoretically, and activities of metabolic enzymes were measured spectrophotometrically. Relative to control animals, STZ-diabetes resulted in a significant 32% decrease in the FM₁ isoform of myosin in EDL and a 24% decrease in IM myosin of SOL. Mass-specific activities of phosphofructokinase, citrate synthase, and cytochrome oxidase were significantly lower in SOL from STZ-diabetic mice than in controls by 23, 18, and 36%, respectively. Intracellular ATP was significantly lower in SOL from STZ-diabetic mice than in controls (3.44±0.20 mol g^–1 wet weight vs. 4.61±0.20 mol g^–1, respectively), as was creatine phosphate (11.98±0.80 mol g^–1 wet weight vs. 14.22±0.44 mol g^–1). In contrast to results from SOL, there were no significant changes in phosphorus metabolites or enzyme activity in EDL. These results show that the effects of IDDM on levels of phosphorus containing metabolites and maximal activities of key regulatory enzymes in muscle are markedly fibertype specific. It is suggested that the muscle type-specific effects of STZ-diabetes may be a consequence of differential accumulation of intracellular fatty acids.     

Comparison of the potentiometric, (31)P NMR, and zero-point titration methods of determining ionized magnesium in erythrocytes

A simple and rapid method of determining ionized magnesium in erythrocytes using a potentiometric clinical analyzer, Microlyte 6 (Kone, Finland), was investigated. The erythrocyte cell membranes were destroyed using ultrasound. The results were compared with those obtained with the (31)P nuclear magnetic resonance spectroscopy method and the zero-point titration method using atomic absorption spectrometry. The results obtained from potentiometry and from the other methods did not differ significantly (Student t test, alpha = 0.01). Total magnesium concentration was determined using atomic absorption spectrometry.     

31P-Nuclear magnetic resonance spectroscopy study of the time course of energy metabolism during exercise and recovery

This study evaluated the time courses of intracellular pH and the metabolism of phosphocreatine (PCr) and inorganic phosphate (P) at the onset of four exercise intensities and recoveries. Non-invasive evaluation of continuous changes in phosphorus metabolites has become possible using³¹P-nuclear magnetic resonance spectroscopy (³¹P-MRS). After measurements at rest, six healthy male subjects performed 4 min of femoral flexion exercise at intensities of 0 (loadless), 10, 20 and 30 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore. Measurements were continuously made during 5 min of recovery. During a series of rest-exercise-recovery procedures,³¹P-MRS were accumulated using 32 scans · spectrum^–1 requiring 12.8 s each. At the onset of exercise, PCr decreased exponentially with a time constant of 27–32 s regardless of the exercise intensity. The time constant PCr resynthesis during recovery was about 27–40 s. The PCr kinetics were independent of exercise intensity. There were similar Pi kinetics at the onset of all types of exercise, while those of Pi recovery became significantly longer at the higher exercise intensities (P < 0.05). Furthermore, the intracellular pH indicated temporary alkalosis just at the onset of exercise, probably due to absorption of hydrogen ions by PCr hydrolysis, and then decrease at a point about 40%–50% of the preexercise PCr. The pH recovery time was longer than that for the P_i or PCr kinetics. By using a more efficient resolution system it was possible to obtain the phosphorus kinetics during exercise and to follow PCr resynthesis within the first few minutes of recovery. From our results it was concluded that in general the time course of PCr and Pi metabolism were unaffected by the exercise intensity, both at the onset of exercise and during recovery, with the exception of P_i recovery.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Triacontanol and Jasmonic Acid Differentially Modulate the Lipid Organization as Evidenced by the Fluorescent Probe Behavior and <Superscript>31</Superscript>P Nuclear Magnetic Resonance Shifts in Model Membranes

Fluorescence resonance energy transfer (FRET), time-resolved fluorescence and anisotropy decays were determined in large unilamellar vesicles (LUVs) of egg phosphatidylcholine with the FRET pair N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine as donor and lissamine rhodamine B 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine as acceptor, using 2-ps pulses from a Ti:sapphire laser on LUVs with incorporated plant growth regulators: triacontanol (TRIA) and jasmonic acid (JA). FRET efficiency, energy transfer rate, rotation correlation time, microviscosity, and diffusion coefficient of lateral diffusion of lipids were calculated from these results. It was observed that TRIA and JA differentially modulated all parameters studied. The effect of JA in such modulations was always partially reversed by TRIA. Also, the generalized polarization of laurdan fluorescence indicated that JA enhances the degree of hydration in lipid bilayers to a larger extent than does TRIA. Solid-state ³¹P magic-angle spinning nuclear magnetic resonance spectra of LUVs showed two chemical shifts, at 0.009 and −11.988 ppm, at low temperatures (20°C), while at increasing temperatures (20–60°C) only one (at −11.988 ppm) was prominent and the other (0.009 ppm) gradually became obscure. However, LUVs with TRIA exhibited only one of the shifts at 0.353 ppm even at lower temperatures and JA did not affect the chemical shifts. An erratum to this article can be found at     

31P nuclear magnetic resonance study on changes in phosphocreatine and the intracellular pH in rat skeletal muscle during exercise at various inspired oxygen contents

We measured ATP, phosphocreatine (PCr), inorganic phosphate (P_i), and the intracellular pH in rat hindlimb muscles during submaximal isometric exercise with various O₂ deliveries using³¹P nuclear magnetic resonance spectroscopy (³¹P NMR) to evaluate changes in energy metabolism in relation to O₂ availability. Delivery of O₂ to muscles was altered by controlling the fractional concentration of inspired oxygen (F _IO₂) at 0.50, 0.28, 0.21, 0.11 and 0.08 with monitoring partial pressure of oxygen and carbon dioxide, and bicarbonate at the femoral artery. The steady-state ratio of PCr : (PCr + P_i) during exercise decreased as a function ofF _IO₂ even at 0.21. Significant acidification of the intracellular pH during exercise occurred at 0.08F _IO₂. Change in the PCr : (PCr + P_i) ratio demonstrated that the oxidative capacity, i.e. the maximal rate of the oxidative phosphorylation reaction, in muscle was not limited by O₂ delivery at 0.50F _IO₂, but was significantly limited at 0.21F _IO₂ or below. Change in the intracellular pH at 0.08F _IO₂ could be interpreted as an increase in lactate, suggesting activation of glycolysis. Correlation between the PCr : (PCr + P_i) ratio and the intracellular pH revealed the existence of a critical PCr : (PCr + P_i) ratio and pH for glycolysis activation at around 0.4 and 6.7, respectively.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

Skeletal muscle: A paradigm for testing principles of bioenergetics

Muscular activity converts chemical energy into useful work and metabolism restores muscle to its original state. This essay explores the organization and interactions of the regulatory system(s) which allow this energy balance to occur. The term energy balance is used in a biochemical rather than a thermodynamic sense—concerned not with deductions from the physical principles of thermodynamics, but rather with those enzymatic processes which nature evolved and which operate at remarkably fixed stoichiometry. Energy balance is a statement of conservation of energy put into biochemical observables.³¹P NMR spectroscopy is one of the most useful techniques for investigating these questions quantitatively under physiological conditionsin vivo. The author (1) describes the rules or principles of biochemical energy balance; (2) discusses sample results from human muscle to demonstrate its use in studying this class of questions; (3) presents a simple model of integrated cellular respiration to demonstrate its sufficiency to account for the main observations.     

Organotin compounds promote the formation of non-lamellar phases in phosphatidylethanolamine membranes

Organotin compounds are important contaminants in the environment. They are membrane active molecules with broad biological toxicity. We have studied the interaction of tri-n-butyltin chloride and tri-n-phenyltin chloride with model membranes composed of different phosphatidylethanolamines using differential scanning calorimetry, X-ray diffraction, ³¹P-nuclear magnetic resonance and infrared spectroscopy. Organotin compounds laterally segregate in phosphatidylethanolamine membranes without affecting the shape and position of the lamellar gel to lamellar liquid-crystalline phase transition thermogram of the phospholipid. This is in contrast with their reported effect on phosphatidylcholine membranes [Chicano et al. (2001) Biochim. Biophys. Acta 1510, 330-341] and emphasises the importance of the nature of the lipid headgroup in determining how the behaviour of lipid molecules is affected by these toxicants. Interestingly, we have found that organotin compounds disrupt the pattern of hydrogen-bonding in the interfacial region of dielaidoylphosphatidylethanolamine membranes and have the ability to promote the formation of hexagonal H_II structures in this system. These results open the possibility that some of the specific toxic effects of organotin compounds might be exerted through the alteration of membrane function produced by their interaction with the lipidic component of the membrane.     

2-O-(2-Aminoethyl)-myo-inositol 1,4,5-trisphosphate as a novel ligand for conjugation: physicochemical properties and synthesis of a new Ins(1,4,5)P(3) affinity matrix

2-O-(2-Aminoethyl)-Ins(1,4,5)P(3), (5), a novel derivative of the Ca(2+)-mobilising second messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], was synthesised from myo-inositol. 5 was found to be a potent mobiliser of intracellular Ca(2+), and an Ins(1,4,5)P(3) affinity matrix synthesised from 5 was effective at selectively binding N-terminal fragments of the Ins(1,4,5)P(3) receptor containing the intact Ins(1,4,5)P(3) binding site. The microprotonation scheme for 5 was resolved and the related constants were determined in comparison with Ins(1,4,5)P(3) and another reactive Ins(1,4,5)P(3) analogue 1-O-(2-aminoethyl-1-phospho)-Ins(4,5)P(2), (2a), by potentiometric and NMR titration methods. The (31)P and (1)H NMR titration curves for compound 5 and Ins(1,4,5)P(3) are remarkably close, indicating analogous acid-base properties and intramolecular interactions for the two compounds. The 1-phosphate-modified Ins(1,4,5)P(3) derivative 2a, on the contrary, behaves as a bisphosphorylated rather than a trisphosphorylated inositol. Thus, 5 is a new reactive Ins(1,4,5)P(3) analogue of considerable potential for investigation of the chemical biology of Ins(1,4,5)P(3)-mediated cellular signalling.     

Effects of gramicidin-A on the adsorption of phospholipids to the air-water interface

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air-water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (H_II) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and ³¹P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the H_II phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature.     

Neurochemical Changes in Brain Induced by Chronic Morphine Treatment: NMR Studies in Thalamus and Somatosensory Cortex of Rats

To investigate the effects of chronic morphine treatment and its cessation on thalamus and the somatosensory cortex, an ex vivo high resolution (500 MHz) ¹H nuclear magnetic resonance spectroscopy (NMRS), in the present study, was applied to detect multiple alterations of neurochemicals and/or neurometabolites in the rats. Ten days of chronic morphine administration was observed to markedly increase the total amount of lactate (Lac), myo-inositol (my-Ins) (each P < 0.01) and aspartate (Asp) (P < 0.05), and significantly decrease that of glutamate (Glu) and glutamine (Gln) in the rats thalamus (each P < 0.05). In the somatosensory cortex, chronic morphine was shown to increase the level of Lac and my-Ins, and decrease that of Glu (each P < 0.05). Interestingly, the ratio of Glu/GABA was found to decrease in these two brain areas after chronic morphine treatment, and among the detectable neurochemicals in those two cerebral areas, only taurine (Tau) showed to result in a significant increment in thalamus during the process of morphine discontinuation (P < 0.05). Moreover, the alterations of multiple neurochemicals due to chronic morphine exhibited a tendency of recovery to the normal level over the course of morphine withdrawal. The results suggested that, in thalamus and the somatosensory cortex, chronic morphine administration and its cessation could induce multiple neurochemical changes, which may involve in the brain energy metabolism, activity and transition of neurotransmitters.     

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing C CPMAS, H, and REDOR solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca²⁺ regulator. ¹³C cross-polarization magic angle spinning spectra of ¹³C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding ¹³C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl ¹³C resonance in the corresponding NMR spectrum. ¹³C-¹⁵N REDOR solid-state NMR spectroscopic experiments revealed the distance between the ¹³C carbonyl carbon of Leu39 and the ¹⁵N amide nitrogen of Leu42 to be 4.2 ± 0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three ²H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11° ± 5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.     

39K nuclear magnetic resonance and a mathematical model of K+ transport in human erythrocytes

³⁹K nuclear magnetic resonance was used to measure the efflux of K⁺ from suspensions of human erythrocytes [red blood cells (RBCs)], that occurred in response to the calcium ionophore, A23187 and calcium ions; the latter activate the Gárdos channel. Signals from the intra- and extracellular populations of ³⁹K⁺ were selected on the basis of their longitudinal relaxation times, T ₁, by using an inversion- recovery pulse sequence with the mixing time, τ₁, chosen to null one or other of the signals. Changes in RBC volume consequent upon efflux of the ions also changed the T ₁ values so a new theory was implemented to obviate a potential artefact in the data analysis. The velocity of the K⁺ efflux mediated by the Gárdos channel was 1.19±0.40 mmol (L RBC)⁻¹ min⁻¹ at 37°C.     

Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes

Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH(*)) are quantified using (15)N nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8 T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290-320 K and at a static magnetic field strength of 14.1 T. Results for ttRNH(*) are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the picosecond-to-nanosecond time scale generally are similar for ttRNH(*) and ecRNH. beta-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH(*) than in ecRNH. Rigidity in the two beta-strands and the glycine-rich region, located along the periphery of the central beta-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH(*) than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of approximately 15 kJ/mol in the apparent activation energies for ttRNH(*) residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between alpha-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.     

NMR analysis of plant nitrogen metabolism

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of ¹H, ²H, ¹³C, ¹⁴N and ¹⁵N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with ²H-, ¹³C- and ¹⁵N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.