Estimation of intracellular sugar phosphate concentrations inSaccharomyces cerevisiae using31P 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, 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.
Analytical Biochemistry, 1999
31 P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved 31 P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum (1 H-31 P HSQC) and 31 P correlation spectroscopy (31 P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts. © 1999 Academic Press 31 P NMR spectroscopy has been shown to be a useful tool for the study of cell metabolism. This is mainly due to the fact that a variety of important metabolites are phosphorylated and that the 31 P nucleus has a 100% natural abundance, thereby eliminating the need of
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.
A fast sensor for in vivo quantification of cytosolic phosphate in Saccharomyces cerevisiae
Biotechnology and Bioengineering, 2015
Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processes which are distributed over different subcellular compartments. Currently, available metabolite measurement protocols allow to measure metabolite whole cell amounts which hinder progress to describe the in vivo dynamics in different compartments, which are driven by compartment specific concentrations. Phosphate (Pi) is an essential component for: (1) the metabolic balance of upper and lower glycolytic flux; (2) Together with ATP and ADP determines the phosphorylation energy. Especially, the cytosolic Pi has a critical role in disregulation of glycolysis in tps1 knockout. Here we developed a method that enables us to monitor the cytosolic Pi concentration in S. cerevisiae using an equilibrium sensor reaction: maltose þ Pi < ¼ > glucose þ glucose-1-phosphate. The required enzyme, maltose phosphorylase from L. sanfranciscensis was overexpressed in S. cerevisiae. With this reaction in place, the cytosolic Pi concentration was obtained from intracellular glucose, G1P and maltose concentrations. The cytosolic Pi concentration was determined in batch and chemostat (D ¼ 0.1 h À 1 ) conditions, which was 17.88 mmol/gDW and 25.02 mmol/gDW, respectively under Pi-excess conditions. Under Pi-limited steady state (D ¼ 0.1 h À 1 ) conditions, the cytosolic Pi concentration dropped to only 17.7% of the cytosolic Pi in Pi-excess condition (4.42 mmol/gDW vs. 25.02 mmol/gDW). In response to a Pi pulse, the cytosolic Pi increased very rapidly, together with the concentration of sugar phosphates. Main sources of the rapid Pi increase are vacuolar Pi (and not the polyPi), as well as Pi uptake from the extracellular space. The temporal increase of cytosolic Pi increases the driving force of GAPDH reaction of the lower glycolytic reactions. The novel cytosol specific Pi concentration measurements provide new insight into the thermodynamic driving force for ATP hydrolysis, GAPDH reaction, and Pi transport over the plasma and vacuolar membranes. specific substrate uptake rate; qCO 2 , the specific carbon dioxide production rate; qO 2 , the oxygen uptake rate; qGly, the specific glycerol production rate; RQ, respiratory quotient. Correspondence to: J. Zhang and S. A. Wahl fax: þ31 (0)15 2782355
31P Nuclear Magnetic Resonance Study of Growth and Dimorphic Transition in Candida albicans
Microbiology, 1983
A 31P NMR study of the fungal pathogen Candida albicans was carried out. Yeast-form cells at different phases of growth, as well as germ tubes and hyphae were examined. In all cases, the NMR spectra showed well separated resonance peaks arising from phosphorus-containing metabolites, the most prominent being attributable to inorganic phosphate (P,) polyphosphates, sugar phosphates and mononucleotides, NAD, ADP and ATP. Relevant signals were also detected in the phosphodiester region. The intensity of most signals, as measured relative to that of PI, was clearly modulated both at the different phases of growth and during yeast-to-mycelium conversion, suggesting significant changes in the intracellular concentration of the corresponding metabolites. In particular, the intensity of the polyphosphate signal was high in exponentially growing, yeast-form cells, then progressively declined in the stationary phase, was very low in germ tubes and, finally, undetectable in hyphae. NMR spectral analysis of the PI region showed that from early-stationary phase, P, was present in two different cellular compartments, probably corresponding to the cytoplasm and the vacuole. From the chemical shift of Pi, the pH values of these two compartments could be evaluated. The cytoplasmic pH was generally slightly lower than neutrality (6.74-8), whereas the vacuolar pH was always markedly more acidic.
Effects of 2-deoxyglucose on Saccharomyces cerevisiae as observed by in vivo 31 P-NMR
FEMS Microbiology Letters, 1989
Saccharomyces cerevisiae cells were treated with 2-deoxyglucose (1 mM) and the effects induced in the levels of phosphorus compounds and in the internal pH were monitored using 31p-NMR. Upon incubation with 2-deoxyglucose a strong decrease in the polyphosphate level was observed and the cytoplasmic pH decreased by about 0.4 units. This shows that 2-deoxyglucose strongly interferes with the cell conditions and consequently, the results of experiments in which 2-deoxyglucose was used to obtain deenergized cells should be carefully reanalysed.
Biotechnology and Bioengineering, 1990
The reg 1 mutation will allow the expression of a cloned gene on a plasmid under the control of a GAL promoter in the presence of glucose. The metabolism of wild-type and reg l mutant strains was examined by in vivo (31)P nuclear magnetic resonance (NMR) spectroscopy. Transient profiles of glucose 6-phosphate, fructose 6-phosphate, fructose 1, 6-diphosphate, and 3-phosphoglycerate indicated that glucose was processed differently for the reg 1 strain despite similar cytoplasrnic pH values and ATP levels. Intracellular phosphate became depleted in the transition to quasi-steady state and limited glycolysis in the reg 1 strain. The glucose uptake step or hexokinase step appears to be altered in the reg 1 strain. The reg 1 strain utilized galactose faster than the wild-type strain under the conditions used for NMR analysis. These results are consistent with the hypothesis that the REG 1 product operates early in the regulatory circuitry for glucose repression. This study illustrates the usefulness of transient information provided by NMR in understanding changes in the metabolism of genetically manipulated organisms.