Labeling metabolic

Metabolic labeling ofproteins with radioactive amino acids... 

Metabolic labeling ofproteins with stable isotopes... 

The use of the in vivo labeling methods described above is limited by the fact that the sample must be grown in the presence of the labeling isotopes. In many cases, it is not feasible to perform in vivo metabolic labeling. For example, for human clinical samples it is not possible to perform in vivo labeling and yet it is highly desirable to obtain accurate quantitative information on protein expression levels within these samples. Therefore, robust methods are needed for quantitation of protein levels in the absence of in vivo labeling with isotopes. 

One day prior to the isolation of polysomes, approximately 5 million HeLa cells are plated into 10-cm dishes. The following day, the media is replaced with fresh DMEM and compound is added to a concentration previously determined to inhibit translation in vivo by 35S-methionine metabolic labeling (see previously). 

To characterize phospholipid oxidation during apoptosis, cellular phospholipids were metabolically labeled at sn-2 position with a natural unsaturated florescent fatty acid containing four conjugated double bonds, cw-parinaric acid (cw-PnA) (Kagan et al, 1998 Tyurina et al, 2001). Oxidative desttuction of any part of the conjugated double bond system of... 

Analytical tools have been developed in order to identify carbohydrate structures as well as carbohydrate-binding proteins and to understand their underlying structure-function relationships of protein-carbohydrate and carbohydrate-carbohydrate interactions lectin arrays [16], glycan microarrays [17, 18], glyco-nanoparticles [19], frontal affinity chromatography [20] and carbohydrate tools for metabolic labeling [21]. 

C and present in the molecule are respectively converted to "CO2 and Its sensitivity depends on the degree of radioactivity of the analyte. Useful for metabolic labelling studies, making metabolites of drugs easy to detect. The detector tends to work better with packed columns since it has a large internal space... 

Sakurai, N., Moriya, K., Suzuki, T., Sofiiku, K, Motiki, H., Nishimura, O., and Utsmni, T. (2007) Detection of co- and post-translational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system. Anal. Biochem. 362, 236-244. 

Metabolic labelling studies on LLO with [2-3H] mannose are carried out in fibroblasts of patients who present with a characteristic CDG-type IIEF transferrin pattern but normal PMM and PMI activities, thereby excluding CDG-Ia and CDG-Ib. Investigations require the extraction and analysis of LLO by HPLC and TLC. 

Metabolic labelling studies require primary skin fibroblasts from patients and healthy controls. It is also helpful to have an internal control from CDG-type I patients, who accumulate shortened LLO, like Man5GlcNAc2 (CDG-Id, CDG-Ie) or Man7GlcNAc2 (CDG-Ig Fig. 4.5.6). 

Fig. 4.5.6 Lipid-linked oligosaccharides (LLO) isolated from different CDG-I patients. Fibroblasts from a control and different CDG-I patients (CDG-lc, CDG-ld, CDG-lg, CDG-li) were metabolically labelled with 2-[3H]mannose for 30 min. [3H]oligosaccharides were released from LLO by mild acid hydrolysis and size-fractionated by HPLC. M5, M7, M9 and G3 refer to the positions of GlcNAc2Man5, GlcNAc2Man7, GlcNAc2Man9 and GlcNAc2Man9Glc3, respectively. The arrow marks shortened [2-3H]mannose-labelled oligosaccharides accumulating in the case of very early CDG-I types like CDG-li, which are often hard to detect by standard LLO-HPLC analysis...

Fig. 4.5.7 TLC analysis of short LLO. Fibroblasts derived from a control (lane 1, left) andaCDG-Ii patient (lane 2, right) were metabolically labelled for 30 min with [2-3H] mannose. Short LLO were extracted with chloroform/methanol (3 2) and further analysis carried out by TLC in a running buffer containing chloroform/methanol/LLO (65 25 4). The position of the origin, and the positions of [3H]ManiGlcNAc2-PP-dolichol and [3H]Man2GlcNAc2-PP-dolichol are indicated...

Metabolic labelling of cells with [2-3H]mannose fails to detect deficiencies in the very early onset of LLO biosynthesis as in CDG-Ij and CDG-Ik (Fig. 4.5.1). In this case,... 

Defects in the early steps of LLO biosynthesis (Fig. 4.5.1) lead to an accumulation of shortened LLO that escape the extraction procedure described above (e.g. CDG-Ik). Moreover, defects in the initiating steps of LLO biosynthesis, such as the addition of the two glucosamine residues and the first mannose residue, are missed by metabolic labelling with [2-3H]mannose. In this case it is necessary to label fibroblasts with [6-3H] glucosamine. 

Metabolic labelling is carried out in primary skin fibroblasts. It is helpful to have an internal control from a CDG-type I patient with an early N-glycosylation deficiency like CDG-Ii or CDG-Ik. 

Metabolically labelled fibroblasts from CDG-patients with defined deficiencies in the very early steps of LLO biosynthesis are compared to LLO profiles of patients with a suspected CDG. 

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... 

Clostridium thermocellum Anaerobe Metabolic labeling Metabolic pathway analysis (175)... 

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