Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction

Abstract

An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented. The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to -35 degrees C, bringing about a fast and complete stop of all metabolic activity. In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching. A liquid-liquid extraction with chloroform at -25 degrees C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels. The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components. The internal standard was recovered to an extent of 85-95%.

Global metabolite analysis of yeast: evaluation of sample preparation methods

“…Yeast cells, like bacterial cells (Jensen et al, 1999;Letisse and Lindley, 2000;Wittmann et al, 2004), are therefore concluded to be sensitive to cold methanol solution either buffered or nonbuffered (Figure 3). Even varying the concentration of cold methanol solution from 25% to 75% did not prevent cell membrane damage as assayed with propidium iodide (data not shown).…”

“…Hans et al (2001) could not detect any amino acids in the supernatant of methanol-quenched yeast cells. In contrast to yeast cells, the intracellular metabolites from bacterial cells leak from the cells when these are brought into contact with cold methanol solution (60% v/v) and several intracellular metabolites have been found in the medium after the quenching process (Jensen et al, 1999;Letisse and Lindley, 2000;Wittmann et al, 2004).…”

“…Their method has recently been applied for intracellular metabolite analysis of microorganisms (Cremin et al, 1995;Ruijter and Visser, 1996;Smits et al, 1998;Jensen et al, 1999;Zaldivar et al, 2002;Maharjan and Ferenci, Sample preparation for global metabolite analysis 1157 2003) and animal tissues (Le Belle et al, 2002). The method presents good recovery of phosphorylated compounds (Smits et al, 1998;Jensen et al, 1999).…”

Hyaluronan Molecular Weight Is Controlled by UDP-N-acetylglucosamine Concentration in Streptococcus zooepidemicus

“…Briefly, 5 ml of cell suspension was centrifuged at 50,000 ϫ g for 2 min at 37°C and subsequently extracted into boiling ethanol. Extracts were enriched using SAX resin columns as described elsewhere (37), except that metabolites were eluted from columns using 2 ml of 0.15 M sodium citrate instead of sodium acetate. Metabolites were analyzed using high-performance anion-exchange chromatography and quantified via an integrated pulsed amperometric detector (36).…”

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea

“…The SPE method for phosphorylated sugars was according to the method described by Jensen et al [17] with slight modification (0.6 mL 2 M ammonium acetate instead of sodium acetate was used to remove the bound phosphorylated sugars from the column) with Isolute SAX 200 mg/ 3 mL SPE column.…”