Abstract
The basic strategy used in most assays of activities involved in the biosynthesis of glycosylphosphatidylinositol (GPI) in mammalian cells is the same as is employed for other lipid biosynthetic pathways. That is, radioactivity is transferred from a water-soluble substrate into a lipophilic product. After the reaction is complete, the differential solubility of the substrate and product(s) is exploited to separate these radiolabeled compounds. In GPI biosynthesis, at least one of the substrates in each step and all of the enzymes in the pathway are membrane-associated and localized to the endoplasmic reticulum. Therefore, multiple GPI biosynthetic activities, as well as some of the substrates for later steps in the pathway, are present in the cellular preparations used in the assays. For this reason, multiple intermediates in GPI biosynthesis are usually generated in a single reaction. Although it is possible to optimize the assay conditions for one step, it is usually impossible to study one reaction independently with this type of cell-free system.
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Stevens, V.L. (1998). In Vitro Analysis of GPI Biosynthesis in Mammalian Cells. In: Gelb, M.H. (eds) Protein Lipidation Protocols. Methods in Molecular Biology, vol 116. Humana Press. https://doi.org/10.1385/1-59259-264-3:1
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DOI: https://doi.org/10.1385/1-59259-264-3:1
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