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Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with gamma-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.

Two isoenzymes of xyloglucan endo-transglycosylase (XET, EC 2.4.1.207) were identified in nasturtium (Tropaeolum majus L.), so far. One is located in seeds (sXET) and is expressed during germination. The other enzyme (eXET) is confined to epicotyls and other growing regions. In this work, we examined catalytic properties of the two XETs and tried to find a correlation with their presumed functions. The two enzymes had similar isoelectric points at about pH 6.5 but had different pH-activity profiles and differed in Km values for xyloglucan-derived oligosaccharides (XGOS) as acceptor substrates. Moreover, they showed clearly distinct preferences in selecting the site of attack on xyloglucan (XG) molecules. While sXET selected the site of cleavage on XG molecules stochastically along the length of their polyglucose main chain and preferred low-molecular mass (MM) XGOS as glycosyl acceptors, eXET attacked the substrate molecule predominantly near the reducing end and showed no preference as to the size of XGOS acceptors. These properties corroborate well with the proposed functions of the two isoenzymes: the sXET plays a role in degrading XG reserves in seeds during germination, whereas the eXET is engaged in cell wall rearrangement and integration of new XG molecules into the preexisting cell wall structure during growth.

Incubation of isolated NaCl-washed cell walls from epicotyls of pea ( Pisum sativum ) and nasturtium ( Tropaeolum majus ) with solutions of various oligosaccharides released among others the cell wall marker enzyme xyloglucan endotransglycosylase (XET, EC 2.4.1.207). The greatest release of XET occurred upon incubation of the cell walls with xyloglucan-derived oligosaccharides (XGOS, DP 7-9). Concomitantly, reduced radioactive nonasaccharide 〚 3 H〛-XLLGol (Gal 2 .Xyl 3 .Glc 3 .〚1- 3 H〛-glucitol) was incorporated into the cell walls. Subsequent hydrolysis of the radioactively labelled cell walls with Trichoderma cellulase liberated XGOS-alditols, DP 7-9 as the sole radioactive products indicating that 〚 3 H〛-XLLGol was incorporated into the cell wall xyloglucan by transglycosylation, as an entity. Oligosaccharides of cello-, chito- and/or oligoglucurono-series were much less effective than XGOS but a substantial liberation of XET and other proteins from plant cell walls could be achieved by the nucleophile 0.1 M imidazole. The specific release of the cell wall-associated XET activity by incubation with xyloglucan-derived oligosaccharides and the simultaneous incorporation of the tritiated xyloglucan nonasaccharide en bloc into the cell walls indicates that XET is present in the cell walls in form of a competent glycosyl-enzyme complex which decomposes by transglycosylation of its glycan moiety to added xyloglucan-oligosaccharide acceptors. This finding suggests a new concept for the regulation of activity of cell wall-associated glycanases/transglycosylases: they exist in plant cell walls in a transiently latent state as covalent glycosyl-enzyme complexes and are active only when suitable glycosyl acceptors become available.