Carbon‐13 nuclear magnetic resonance spectroscopy of living cells and their metabolism of a specifically labeled <sup>13</sup>C substrate

Eakin, Robert E.; Morgan, L. O.; Gregg, Charles T.; Matwiyoff, Nicholas A.

doi:10.1016/0014-5793(72)80726-9

Cited by 114 publications

(61 citation statements)

References 21 publications

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“…l3C NMR had previously been used to delineate biosynthetic pathways. However, these studies either were restricted to monitoring the utilization of substrates and accumulation of end produces or made use of the conventional acid extracts of cells to detect 13C-labeled intermediary metabolites (3)(4)(5)(6). Our recent work on suspensions of E. coli cells (2) showed that in addition to end products of glucose metabolism the resonances of particular carbons in transient intermediates can be detected in spectra accumulated for 1 min, and the concentrations of these intermediates can be followed as a function of time.…”

mentioning

confidence: 99%

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

Hollander

Uğurbil

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

116

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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.

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confidence: 99%

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

Hollander

Uğurbil

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

116

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“…End-product enrichment by the use of labeled 13C compounds has been reported. When 13C-enriched glucose was fed to yeast, two weak, barely resolved resonances from end products of glycolysis were detected by 13C NMR (6). More recently, 13C spectra of intact soybean ovules (7) and erythrocytes (8) exposed to '3CO2 have been reported and some long-lived compounds have been identified.…”

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confidence: 99%

High-resolution 13C nuclear magnetic resonance studies of glucose metabolism in Escherichia coli.

Uğurbil¹,

Tr²,

Hollander³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

128

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High-resolution 13C nuclear magnetic resonance spectra of suspensions of Escherichia coli cells have been obtained at 90.5 MHz by using the Fourier transform mode. Anaerobic cells incubated with 11-'3Cjglucose show a time course of glycolysis in which the a and P glucose anomers disappear at different rates, lactate, succinate, acetate, alanine, and valine accumulate as end products of glycolysis, and fructose bisphosphate appears as an intermediate. It is shown that fructose bis osphate is labeled at C-1 and C-6Eduring [1-13C glucose catablism. Upon oxygenation, glutamate appears with the 13C enrichment at the CA, C-3, and C-2 positions, with the CA most intense. From the position of the 13C label we conclude that valine is formed by condensation of pyruvate and that carbon enters the tricarboxylic acid cycle mainly through acetyl CoA.31P high-resolution nuclear magnetic resonance (NMR) studies of cellular suspensions and of intact tissue have shown-how this noninvasive technique can follow concentrations and kinetics of metabolites in vivo (1-3). Because several phosphate metabolites have chemical shifts that are sensitive to pH under physiological conditions, it has also been possible to distinguish intracellular and extracellular pH values (2-5). Although 31P NMR has been valuable in studying cellular problems, it is restricted to monitoring phosphorylated metabolites.In the present report we show that it is possible to observe well-resolved 13C NMR of numerous metabolites in anaerobic and aerobic suspensions of Escherichia coli cells and to assign certain resonances to particular carbons of transient intermediates as well as of end products. End-product enrichment by the use of labeled 13C compounds has been reported. When 13C-enriched glucose was fed to yeast, two weak, barely resolved resonances from end products of glycolysis were detected by 13C NMR (6). More recently, 13C spectra of intact soybean ovules (7) and erythrocytes (8) exposed to '3CO2 have been reported and some long-lived compounds have been identified.Biosynthetic pathways have been followed by feeding various 13C-labeled substrates to different microorganisms and measuring the 1'3C NMR spectra of extracts (9-12). 13C NMR spectra of labeled macromolecules have been obtained in whole cells before extraction (13,14). Recently, glucose utilization was measured by natural-abundance 13C NMR in suspensions of acetone-treated yeast cells in which the cell membranes were rendered permeable and the rate of glycolysis was decreased -100-fold, thus allowing long accumulation times (15).In the present study we used 13C-enriched glucose and a high-frequency 13C NMR spectrometer to monitor metabolism in anaerobic and aerobic Escherichia coli suspensions. We followed the time course of glucose metabolism in well-resolved spectra obtained with 1-min accumulations and identified intermediates as well as end products. Furthermore, we observed that more than one position on several intermediates were labeled with '3C during catabolism of glucose enr...

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“…Experimental techniques using 13 C-enrichment and high-resolution NMR have been performed in metabolic studies carried out on living cells and organs of mammals. [33][34][35] For example, studies on the last two steps of the MEP pathway have demonstrated that the metabolism of 13 C-enriched substrates and direct 13 C-NMR measurement of the cell lysate revealed the products from enzyme reactions. 36,37) This approach also provides a new strategy to determine the function of unknown biosynthetic genes.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Total Synthesis of Gibberellin A₄from Acetate

Sugai

Miyazaki

Mukai

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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Carbon‐13 nuclear magnetic resonance spectroscopy of living cells and their metabolism of a specifically labeled ¹³C substrate

Cited by 114 publications

References 21 publications

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

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

High-resolution 13C nuclear magnetic resonance studies of glucose metabolism in Escherichia coli.

Enzymatic Total Synthesis of Gibberellin A₄from Acetate

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Carbon‐13 nuclear magnetic resonance spectroscopy of living cells and their metabolism of a specifically labeled 13C substrate

Cited by 114 publications

References 21 publications

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

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

High-resolution 13C nuclear magnetic resonance studies of glucose metabolism in Escherichia coli.

Enzymatic Total Synthesis of Gibberellin A4from Acetate

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Carbon‐13 nuclear magnetic resonance spectroscopy of living cells and their metabolism of a specifically labeled ¹³C substrate

Enzymatic Total Synthesis of Gibberellin A₄from Acetate