Metabolic labeling of cells with

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,... 

Depending on the number of phosphorylation sites in the phosphoprotein to be analyzed, and the efficiency of proteolytic digestion as few as a 100 Cerenkov cpm of a phosphoprotein may suffice to give a satisfactory analytical peptide map. Ideally, a few hundred counts per minute should be used, and for most phosphoproteins this should be attainable by metabolic labeling of cells with and easily achieved if in vitro phosphorylation with [y- P]ATP is used to label the protein. If phospho-amino acid determination, secondary digestion, or Edman degradation are to be carried out on individual phosphopeptides (Sections I-L), then commensurately more starting radioactivity will be required. 

Methods to detect and characterize cellular protein myristoylation and palmitoylation are invaluable in cell biology, immunology, and virology. Recently, we developed co-alkynyl fatty acid probes for monitoring myristoylation and palmitoylation of cellular proteins. This article describes a biochemical procedure for metabolic labeling of cells with co-alkynyl fatty acids and click chemistry. 

Analysis of the primary protein structure of the human 5-HT1B receptor reveals a putative site for palmitoylation, i.e., a cysteine residue located in the short carboxyl tail of the receptor (141). A recombinant c-myc epitope-tagged 5-HT1B receptor was expressed in Sf9 insect cells and palmitoylation of the receptor was demonstrated by metabolic labeling of the cells with [3H]palmitic acid. 

Amino acid sequence analysis of the human 5-HT1B receptor has revealed consensus phosphorylation sites in all intracellular loops for PKA and PKC. Phosphorylation of the 5-HT1B receptor was demonstrated by metabolic labeling of the 5-HTib receptor-expressing Sf9 cells with [32Pi]phosphate (141). This posttranslational modification was proposed to be involved in receptor regulation such as desensitization. 

There are two main procedures for measuring PI 3-kinase activity which measure lipid kinase activity in intact cells or broken cell lysates respectively, and both rely on detecting the transfer of the y- phosphate of ATP to the D-3 position of the inositol head group of phosphoinositide lipids. The first method relies on metabolic labeling of intact cellular pools of ATP with [32P]Pi followed by lipid extraction (3,4) and separation of the phosphorylated lipids by high-performance liquid chromatography (HPLC) analysis (5). The advantages of this procedure are ... 

Another widely used approach to the elucidation of metabolic sequences is to feed cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard radioactive isotopes, such as and stable heavy isotopes, such as or (Table 18.3). Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable tags for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13). 

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