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

Abstract

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...

Ultrahigh field magnetic resonance imaging and spectroscopy

“…The label is then transferred into the amino acids glutamate, aspartate and glutamine. The ability to monitor the labeling of intracellular compounds in intact cells was first demonstrated using E. coli [116]. Today, such highly specific data can be obtained in human and animal brains from relatively small volume elements.…”

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

“…Most of these studies have used 31P NMR to obtain information on the concentration, kinetics, and physical state of phosphorylated metabolites in vivo and to measure intracellular pH. By using the techniques developed for the 31P NMR studies of intact cells, it has also been possible to observe well-resolved NMR signals from 13C nuclei in suspensions of Escherichia coli cells during catabolism of [1-'3C]glucose (2), and in rat liver cells during gluconeogenesis in the presence of [1,3-13C]glycerol and [3-13C]alanine (1). l3C NMR had previously been used to delineate biosynthetic pathways.…”

“…Bubbling provided continuous stirring of the samples and prevented the accumulation of CO2/HCO%, which is generated during glycolysis. (2). The C1 resonance of aFru-P2 is separable from that of fFru-P2 in the intact cell spectrum and is assigned in Fig.…”

“…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. Furthermore, it was shown that several of the intermediates, and especially fructose 1,6-bisphosphate (Fru-P2), were labeled at more than one position due to scrambling of the label initially introduced as [1-13C]glucose.…”

NMR measurement of brain oxidative metabolism in monkeys using 13C‐labeled glucose without a 13C radiofrequency channel

“…NMR studies of metabolism with the use of 13 C-labeled glucose have considerably evolved over the past 25 years, following pioneer work on cell suspensions (1,2). Since the sensitivity of 13 C spectroscopy is intrinsically low, investigators performed the first in vivo brain studies using indirect { 13 C}-1 H detection of amino-acid labeling, to take advantage of the higher sensitivity of 1 H (3,4).…”