Metabolic labeling culture

It is postulated that inhibition of PtdCho synthesis and the release of choline are key steps associated with excitotoxicity and are common to NMDA and AMPA receptor stimulation. The mechanism of inhibition of PtdCho is not fully understood. Metabolic labeling experiments in cortical cultures demonstrate that NMDA receptor over activation does not modify the activity of phosphochohne or phospho-ethanolamine cytidylyltransferases but strongly inhibits choline and ethanolamine phosphotransferase activities. This effect is observed well before any significant membrane damage and cell death. Moreover, cholinephosphotransferase activity is lower in microsomes from NMDA-treated cells. These results show that membrane... 

Metabolic Labeling Pattern of Glycosphinqolipids in Familial Hypercholesterolemic Heterozygous Fibroblasts and Co-cultured Normal and Familial Hypercholesterolemic Homozygous Fibroblasts... 

Metabolic labeling is an in situ method which employs or isotope-encoded ( C or H) amino acids. Organisms are grown in controlled media under defined conditions in order to ensure translation of mRNA entities to uniformly isotopically encoded proteins. Consequently, the approach is generally restricted to cell culture systems and is not applicable to quantitative MS analysis of proteins derived from... 

One advantage of the small size of a-factor and related pheromones is that they can be directly visualized after metabolic labeling. Few other proteins are so small, thus S. cerevisiae a-factor and related proteins like AFRP, as well as pheromones from other species, are often the only proteins that migrate near the dye front in SDS-PAGE analysis, and are far away from the bulk of larger cellular proteins [91,92]. In addition, radiolabeling is assured, as all prenylated molecules have at least one Cys residue in the CAAX motif that can be radiolabeled by S-Cys. Thus, direct visualization from protein preparations of whole cell extracts or culture supernatants is feasible. 

Radioactive labeling. Tumor cells can be metabolically labeled with radioactive agents by incubating with H-thymidine (1-5 /xCi/ml) in complete culture medium and labeling for 18-24 h. At the end of... 

These results leave an element of uncertainty because miuntenance of constant specific radioactivity on recrystallization is not an infallible criterion of radioactive purity. The authors were unable to find a system of paper chromatography that separated glutamic acid from a-aminoadipic acid. Added to this is the fact that Schweet et al. (10) could find no radioactivity in a-aminoadipic acid added as a trapping agent to cultures of Neurospora metabolizing labeled ly ne, and Suda et al. (169) found no formation of this compound in a bacterial enzyme system that oxidized Ijrsine to 5-aminovaleric acid. 

The first reports of metabolic labeling appeared in 1999. In this procedure, yeast cells were grown in two media, one of which used N-emiehed media. " The two yeast cultures were combined and the proteins of interest were digested with trypsin before mass speetrometry analysis. The labeling by use of an N-ammonium salt results in the eomplete labeling of amino acid and could be quantified with mass spectrometry. The corresponding mass shift between the unlabeled and the labeled form of the peptide requires high resolution mass spectrometry for analysis. 

If only very small amounts of material are available (this is often the case with metabolically-labelled glycopeptides from cell cultures) and the sample weight cannot be determined accurately it is usually adequate to incubate the material overnight with 2.5 mg/ml pronase. Thereafter the procedure could follow that described above or, since only very small quantitites of protein are present in the sample, the pronase could be inactivated by heating at 100°C for 10 min and samples applied directly to lectin affinity columns. 

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