Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

Advanced Nuclear Magnetic Resonance

Transparencias en inglés de la asignatura "Resonancia Magnética Nuclear Avanzada" (Advanced Nuclear Magnetic Resonance) (36643) que se imparte en el Máster de Química Médica como asignatura optativa de 3 créditos ECTS. En esta asignatura se completa el estudio iniciado en la asignatura de quinto curso de la licenciatura en Química "Determinación estructural" (7448) y en la del Grado de Química de tercer curso "Determinación estructural de los compuestos orgánicos" (26030) en lo referente a técnicas bidimensionales de resonancia magnética nuclear. Además se proporcionan los conocimientos necesarios para poder interpretar RMN de otros núcleos activos en RMN no estudiados hasta el momento como 19F, 31P, 2H, 28Si y 15N así como sus acoplamientos con los núcleos de 1H y 13C.

Nuclear magnetic resonance saturation at intermediate values of H, the RF magnetic field.

"Contract N-onr-401-15."

Nuclear magnetic resonance: applications to organic chemistry

Mode of access: Internet.

The use of simultaneous H-1 & P-31 magic angle spinning nuclear magnetic resonance measurements to characterize energy metabolism during the conversion of muscle to meat

In order to investigate the potential of magic angle spinning nuclear magnetic resonance (MAS NMR) in the elucidation of post-mortem metabolism in muscle biopsies, simultaneous H-1 and (31)p MAS NMR measurements were made continuously on postmortem (20 min to 24 h) muscle longissimus samples from rabbits. The animals had either been or not been given adrenaline (0.5 mg kg(-1) 4 h pre-slaughter) to deplete stores of muscle glycogen. The intracellular pH was calculated from H-1 spectra, and the post-mortem rate of formation of lactate was followed and quantified. Comparison of measurements made on muscle samples from rabbits treated with adrenaline with measurements made on muscle samples from untreated' rabbits revealed significant effects of adrenaline treatment on both pH (pH24 h = 6.42 vs. pH24 It = 5.60) and formation of lactate (16 mmol g(-1) vs. 65 mmol g(-1)). The P-31 NMR spectra were used to follow the rate of degradation of ATP and phosphocreatine. The present study clearly shows that MAS NMR has potential for the study of post-mortem energy metabolism.

Salt diffusion and distribution in meat studied by Na-23 nuclear magnetic resonance imaging and relaxometry

This study introduces the use of combined Na-23 magnetic resonance imaging (MRI) and Na-23 NMR relaxometry for the study of meat curing. The diffusion of sodium ions into the meat was measured using Na-23 MRI on a 1 kg meat sample brined in 10% w/w NaCl for 3-100 h. Calculations revealed a diffusion coefficient of 1 x 10(-5) cm(2)/s after 3 h of curing and subsequently decreasing to 8 x 10(-6) cm(2)/s at longer curing times, suggesting that changes occur in the microscopic structure of the meat during curing. The microscopic mobility and distribution of sodium was measured using Na-23 relaxometry. Two sodium populations were observed, and with increasing length of curing time the relaxation times of these changed, reflecting a salt-induced swelling and increase in myofibrillar pore sizes. Accordingly, the present study demonstrated that pore size and thereby salt-induced swelling in meat can be assessed using Na-23 relaxometry.

Evaluation of the sonically induced narrowing of nuclear magnetic resonance spectra of solids

The Nuclear Magnetic Resonance (NMR) spectra of liquids contain a wealth of quantitative information that may be derived, for instance, from chemical shifts and spin-spin couplings. The available information depends on the incoherent rapid molecular motion that causes complicating effects present in the solid state to average to zero. Whereas liquid state NMR spectra show narrow lines, the corresponding NMR spectra from the solid state are normally composed of exceedingly broad resonance lines due to highly restricted molecular motion. It is, therefore, difficult to obtain directly as detailed information from the spectra of solids as from those derived from the liquid state. Studies on a new technique (SINNMR, the sonically induced narrowing of the NMR spectra of solids) to remove line broadening effects in the NMR spectra of the solid state are reported within this thesis. SINNMR involves narrowing the NMR absorptions from solid particles by irradiating them with ultrasound when they are suspended in a support liquid. It is proposed that ultrasound induces incoherent motion of the suspended particles, producing motional characteristics of the particles similar to those of rather large molecules. The first report of apparently successful experiments involving SINNMR[1] emphasised both the irreproducibility of the technique and the uncertainty regarding its true origin. If SINNMR can be made reproducible and the effect definitively attributed to the sonically induced incoherent motional averaging of particles, the technique could offer a simple alternative to the now classical magic-angle spinning (MAS) NMR[2] and the recently reported dynamic angle spinning (DAS)[3] and double rotation (DOR)[4] techniques. Evidence is presented in this thesis to support the proposal that ultrasound may be used to narrow the NMR spectral resonances from solids by inducing incoherent motion of particles suspended in support liquids and, additionally, for some solids, by inducing rotational motion of molecular constituents in the lattices of solids. Successful SINNMR line narrowing using 20 kHz ultrasound is reported for a variety of samples: including trisodium orthophosphate, polytetrafluoroethylene and aluminium alloys. Investigations of SINNMR line narrowing in trisodium phosphate have revealed the relationship between ultrasonic power, particle size and support liquid density for the production of optimum SINNMR conditions. It is also proposed that the incoherent motion of particles induced by 20 kHz ultrasound can originate from interactions between acoustically induced cavitation microjets and particles.

Nuclear magnetic resonance spectroscopy and ultrasound

The work described in this thesis is directed to the examination of the hypothesis that ultrasound may be used to perturb molecular motion in the liquid phase. These changes can then be detected by nuclear magnetic resonance (NMR) in spin-lattice and spin-spin relaxation times. The objective being to develop a method capable of reducing the pulsed NMR acquisition times of slowly relaxing nuclei. The thesis describes the theoretical principles underlying both NMR spectroscopy and ultrasonics with particular attention being paid to factors that impinge on testing the above hypothesis. Apparatus has been constructed to enable ultrasound at frequencies between 1 and 10 mega-hertz with a variable power up to 100W/cm-2 to be introduced in the NMR sample. A broadband high frequency generator is used to drive PZT piezo-electric transducer via various transducer to liquid coupling arrangements. A commercial instrument of 20 kilo-hertz has also been employed to test the above hypothesis and also to demonstrate the usefulness of ultrasound in sonochemistry. The latter objective being, detection of radical formation in monomer and polymer ultrasonic degradation. The principle features of the results obtained are: Ultrasonic perturbation of T1 is far smaller for pure liquids than is for mixtures. The effects appear to be greater on protons (1H) than on carbon-13 nuclei (13C) relaxation times. The observed effect of ultrasonics is not due to temperature changes in the sample. As the power applied to the transducer is progressively increased T1 decreases to a minimum and then increases. The T1's of the same nuclei in different functional groups are influenced to different extents by ultrasound. Studies of the 14N resonances from an equimolar mixture of N, N-dimethylformamide and deuterated chloroform with ultrasonic frequencies at 1.115, 6, 6.42 and 10 MHz show that as the frequency is increased the NMR signal to noise ratio decreases to zero at the Larmor frequency of 6.42 MHz and then again rises. This reveals the surprising indication that an effect corresponding to nuclear acoustic saturation in the liquid may be observable. Ultrasonic irradiation of acidified ammonium chloride solution at and around 6.42 MHz appears to cause distinctive changes in the proton-nitrogen J coupling resonance at 89.56 MHz. Ultrasonic irradiation of N, N-dimethylacetamide at 2 KHz using the lowest stable power revealed the onset of coalescence in the proton spectrum. The corresponding effect achieved by direct heating required a temperature rise of approximately 30oC. The effects of low frequency (20 KHz) on relaxation times appear to be nil. Detection of radical formation proved difficult but is still regarded as the principle route for monomer and polymer degradation. The initial hypothesis is considered proven with the results showing significant changes in the mega-hertz region and none at 20 KHz.

Nuclear magnetic resonance studies of molecular interactions and structures

This thesis is concerned with the investigation, by nuclear magnetic resonance spectroscopy, of the molecular interactions occurring in mixtures of benzene and cyclohexane to which either chloroform or deutero-chloroform has been added. The effect of the added polar molecule on the liquid structure has been studied using spin-lattice relaxation time, 1H chemical shift, and nuclear Overhauser effect measurements. The main purpose of the work has been to validate a model for molecular interaction involving local ordering of benzene around chloroform. A chemical method for removing dissolved oxygen from samples has been developed to encompass a number of types of sample, including quantitative mixtures, and its supremacy over conventional deoxygenation technique is shown. A set of spectrometer conditions, the use of which produces the minimal variation in peak height in the steady state, is presented. To separate the general diluting effects of deutero-chloroform from its effects due to the production of local order a series of mixtures involving carbon tetrachloride, instead of deutero-chloroform, have been used as non-interacting references. The effect of molecular interaction is shown to be explainable using a solvation model, whilst an approach involving 1:1 complex formation is shown not to account for the observations. It is calculated that each solvation shell, based on deutero-chloroform, contains about twelve molecules of benzene or cyclohexane. The equations produced to account for the T1 variations have been adapted to account for the 1H chemical shift variations in the same system. The shift measurements are shown to substantiate the solvent cage model with a cage capacity of twelve molecules around each chloroform molecule. Nuclear Overhauser effect data have been analysed quantitatively in a manner consistent with the solvation model. The results show that discrete shells only exist when the mole fraction of deutero-chloroform is below about 0.08.