13C nuclear magnetic resonance studies of anaerobic glycolysis in suspensions of yeast cells.

Abstract

Anaerobic glycolysis in Saccharomyces cerevisiae has been studied by 13C NMR at 90. (7), was grown to one-fourth of saturation at 30°C in a liquid medium containing, per liter, 10 g of Bacto-peptone, 5 g of yeast extract, 4 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4, 0.5 g of CaC12, and 30 g of glucose.Prior to harvesting, the cultures were cooled to 5°C in icecold water with continuous shaking. Subsequently, the cells were collected by low-speed centrifugation at 4°C and washed twice in the ice-cold suspension medium, and the pellet was resuspended in an equal volume of medium. The resuspension medium contained 0.85 g of KH2PO4, 0.15 g of K2HPO4, 0.5 g of MgSO4, and 0.1 g of NaCl per liter and, unless otherwise noted, 50 mM pyrophosphate as buffer (pH adjusted to 6 with NaOH). The final yeast suspension was kept in an ice bath until used.1'3C NMR spectra at 90.52 MHz were obtained by using a Bruker HX-360 NMR spectrometer. The spectra were accumulated in 1-min blocks (450 pulse, 0.34-sec repetition time, 200 scans) and stored.continuously on a disk. NMR samples (10 mm sample tube diameter) contained 2 ml of the yeast suspension. The sample was warmed to 20°C and a 95% N2/5% CO2 gas mixture was bubbled through the suspension at 10 cm3/min during the time of the experiment. Bubbling provided continuous stirring of the samples and prevented the accumulation of CO2/HCO%, which is generated during glycolysis. Fig. 1. The spectrum is the sum of 200 free induction decays accumulated in t1 min, between 9 and 10 min after glucose Abbreviations: Fru-P2, fructose 1,6-bisphosphate; TPI, triosephosphate isomerase.

In vivo 13C NMR spectroscopy and metabolic modeling in the brain: a practical perspective

“…2B) [2,4] h-hydroxybutyrate [20]. Modeling with nonglucose substrates is complicated by the fact that, in contrast to glucose, the rate of uptake of 13 C-label into brain metabolism is dependent on the substrate concentration in the blood.…”

“…The potential of 13 C NMR spectroscopy to study metabolic pathways was demonstrated using suspensions of microorganisms [1,2]. The first attempt to model the flow of 13 C label into the TCA cycle was made in 1983 by Chance et al [3] in the heart, at a time when such analysis required some of the fastest computers available.…”

“…We have chosen to focus in this review on metabolic modeling which uses a one-compartment model and [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose or [1,[6][7][8][9][10][11][12][13] C 2 ]glucose as a metabolic substrate. Other tracers and more complex metabolic models can be used, such as two-compartment (neuron-astrocyte) models.…”

Observation by 13C NMR of metabolites in differentiating amoeba

“…The selectivity and resolution of the method with respect to the chemical nature of cellular components renders it attractive for studies of cell physiology [ 8,9].…”

Progress towards in vivo brain 13C-MRS in mice: Metabolic flux analysis in small tissue volumes

“…Glucose metabolism was the first to be examined with 13 C MRS experiments in yeast [118] and E. coli [122], the latter showing time courses of by-products related to glucose catabolism. Few years later a first attempt of modelling glucose uptake was made on data collected from yeast cells fed with [1e 13 C] and [6e 13 C] glucose with a Michaelis-Menten kinetic model [123], while main chemical reactions of the TCA cycle were estimated for the first time in cardiac tissue in vitro [11]. Up to this point, computational methods were still immature and magnetic fields available for research reached approximately 2 Tesla.…”