Identification and Quantitation of Phosphorus Metabolites in Yeast Neutral pH Extracts by Nuclear Magnetic Resonance Spectroscopy (original) (raw)
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Biotechnology and Bioengineering, 1990
Subcellular compartments, such as the vacuole in yeast, play important roles in cell metabolism and in cell response to external conditions. Concentrations of inorganic phosphate and pH values of the vacuole and cytoplasm were determined for anaerobic Saccharomyces cerevisiae cells based upon 31P NMR spectroscopy. A new approach allows the determination of these values for the vacuole in cases when the resonance for inorganic phosphate in the cytoplasm overlaps with the resonance for inorganic phosphate in the vacuole. The intracellular inorganic phosphate resonance was first decomposed into two components by computer analysis. The assignments of the components were determined from in vivo correlations of Pi chemical shift and the chemical shifts of the cytoplasmic sugar phosphates, and the pH dependency of the resonance of pyrophosphate and the terminal phosphate of polyphosphate (PP,) which reside in the vacuole. An in vivo correlation relating PP, and PYc chemical shifts was established from numerous evaluations of intracellular compositions for several strains of S. cerevisiae. This correlation will aid future analysis of 31P NMR spectra of yeast and will extend NMR studies of compartmentation to cellular suspensions in phosphate-containing medium. Application of this method shows that both vacuolar and extracellular Pi were phosphate reserves during glycolysis in anaerobic S. cerevisiae. Net transport of inorganic phosphate across the vacuolar membrance was not correlated with the pH gradient across the membrane.
Biotechnology and Bioengineering, 1988
A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar ghosphates in Saccharomyces cerevisiae based upon P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the 31P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivo correlations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, p-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly labelled compounds, and has the advantage that other important metabolic state variables such as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same 31P spectrum. Extension of this strategy to other cellular systems should be straightforward.
Biotechnology and Bioengineering, 1990
Estimation of intracellular intermediary metabolite levels is of fundamental importance to characterize cell metabolic processes and their regulation. Usually, intracellular intermediates are determined by stopping the cell metabolism, e.g., by immersing the cell sample in liquid nitrogen, and performing percNoric acid extracts of the cells. The metabolite levels are then obtained either by standard analytical methods'-3 or by using nuclear magnetic resonance (NMR) ~pectroscopy.~*~ Using this technique, it is possible to obtain only one point per sample because the method is destructive. Thus, transient studies are not possible on the same cell sample, and a series of aliquots are required with the assumption that all have the same dynamic behavior and are exposed to the same initial conditions. Also, information on compartmentalization within the cell is lost when extracts are prepared. For example, this prevents differentiation of compounds in the cytoplasm from those in the vacuole in yeast.
Phosphorus NMR and Its Application to Metabolomics
Analytical Chemistry
Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. 13 C-carbon and 15 N-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike 13 C and 15 N, 31 Pphosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, 31 P NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but 31 P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating 31 P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of 31 P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of
Combining solid-state and solution-state 31P NMR to study in vivo phosphorus metabolism
Proceedings of the National Academy of Sciences, 1985
Otherwise unavailable information concerning the distribution of phosphorylated compounds in biological systems is obtained by a combined solid-state/solution-state NMR approach, illustrated here for oocytes from Rana pipiens. General methodology is developed, and further extensions are proposed. The following conclusions pertain to the specific system under examination. (i) Nucleoside phosphates can be observed by magic-angle sample spinning of the lyophilized material. (ii) The solid-state NMR technique of dipolar decoupling provides no additional resolution of the phospholipid and phosphoprotein components of the yolk. However, cellular death produces sufficient pH changes to cause the phospholipid and protein phosphate peaks to become resolvable. The concentration of nucleoside phosphates also decreases. (iii) The phospholipid and phosphoprotein components are shown by computer simulation to be present in a ratio of 40:60, respectively. (iv) The amounts of inorganic phosphate, n...
Biochemistry, 1987
Simultaneous 23Na and 31P N M R spectra were obtained from a number of yeast suspensions. Prior to N M R spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they Lad no polyphosphate species visible in the 31P N M R spectrum. In the N M R experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na', and a shift reagent for the Na+ N M R signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The N M R spectra revealed that the cells always lost N a + and gained phosphorus. However, the nature of the N a + efflux time course and the P metabolism differed depending
Applied and environmental microbiology, 1995
The proposed pH buffering and phosphagenic functions of polyphosphate were investigated by subjecting chemostat-cultivated Saccharomyces cerevisiae to alkalinization (NaOH addition) and anaerobiosis. The subsequent changes in intracellular phosphate-containing species were observed in situ by nuclear magnetic resonance (NMR) spectroscopy by using the NMR cultivator we developed. For the alkalinization experiments, changes in catabolite secretion were also measured in parallel experiments. Additionally, a range of potential neutralization capacity was investigated: a dilute culture and concentrated cultures with low or high polyphosphate content. The concentrated cultures displayed increased cytosolic pH and rapid polyphosphate degradation to small chains. The pH changes and extent of polyphosphate degradation depended inversely on initial polyphosphate content. The dilute culture restored extracellular pH rapidly and secreted acetate. The concentrated culture with low polyphosphate ...
Analytical and Bioanalytical Chemistry, 2016
The study aim was to unambiguously assign nucleotide sugars, mainly UDP-X that are known to be important in glycosylation processes as sugar donors, and glucosephosphates that are important intermediate metabolites for storage and transfer of energy directly in spectra of intact cells, as well as in skeletal muscle biopsies by 1 H high-resolution magic-angle-spinning (HR-MAS) NMR. The results demonstrate that sugar phosphates can be determined quickly and non-destructively in cells and biopsies by HR-MAS, which may prove valuable considering the importance of phosphate sugars in cell metabolism for nucleic acid synthesis. As proof of principle, an example of phosphate-sugar reaction and degradation kinetics after unfreezing the sample is shown for a cardiac muscle, suggesting the possibility to follow by HR-MAS NMR some metabolic pathways.
The metabolic state of the rat liver in vivo measured by 31P-NMR spectroscopy
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1986
Previous 3tp nuclear magnetic resonance (NMR) studies have measured the concentrations of phosphates, free Mg 2+, pH and flux through enzyme-catalyzed reactions in a variety of tissues. A surgically-implanted coil has been developed to measure these parameters in the rat liver in vivo, and to assess the effect of external perturbations on the concentrations and physiological environment of phosphorus metabolites in the liver. The sensitive volume and optimal pulse were determined for the coil, which was insulated to exclude signal from surrounding tissues. The metabolic stability of the liver during acquisition of spectra was demonstrated by normal values for [Pi ], [ATP], [iactatel, and [pyruvate] in livers which were freeze-clamped immediately after completion of the NMR experiment. The stability was also confirmed by constant values for intracellular pH (7.2), free [Mg 2+ ] (0.7 filM), and NMR detectable [Pi]/[ATP]. The sensitivity of the 31P-NMR spectrum of the liver in vivo to the physiological state of the animals was illustrated by comparing spectra from fed and 48 h fasted rats. The major qualitative differences were an increase in the pyridine nucleotide/adenine nucleotide ratio, and a small, but consistent shift in the frequency of the composite phosphomonoester peak. The spin-lattice relaxation time of each major phosphate resonance was measured in vivo using a modified homospoii saturation recovery pulse sequence; the T I of ATP ~,-phosphate was 0.17 s. Selective saturation experiments did not detect magnetization transfer between the ATP y-phosphate and inorganic phosphate.