Dataset Information

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix.

ABSTRACT:

Introduction

Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.

Objectives

To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions.

Methods

We present a double-labelling LC-MS approach with a ¹³C-labeled yeast cellular extract as complex surrogate matrix, and ¹³C¹⁵N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates.

Results

While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions.

Conclusions

Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study.

Graphical abstract

SUBMITTER: Gil A

PROVIDER: S-EPMC5126204 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix.

Gil Andres A Siegel David D Bonsing-Vedelaar Silke S Permentier Hjalmar H Reijngoud Dirk-Jan DJ Dekker Frank F Bischoff Rainer R

Metabolomics : Official journal of the Metabolomic Society 20161128 1

<h4>Introduction</h4>Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed.<h4>Objectives</h4>To study how a complex cellular matrix extracted from yeast (<i>Saccharomyces cerevisiae</i>) may affect the degradation profiles of nucleotide triphosphates extracted under ...[more]

PMID: 27980501

Similar Datasets

Project description:A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the full variety of indigenous enzymatic activity of the activated sludge source material could not be restored, experimental modifications, e.g. different lysate filtration, significantly enhanced specific enzyme activities (e.g. >96% removal of the antibiotic erythromycin). Therefore, the approach presented in this study provides the experimental basis for a further elucidation of the enzymatic processes underlying wastewater treatment on the level of native proteins.

Dataset Information

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix.

Introduction

Objectives

Methods

Results

Conclusions

Graphical abstract

Publications

The degradation of nucleotide triphosphates extracted under boiling ethanol conditions is prevented by the yeast cellular matrix.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets