E-Book Overview
An all-star collection of readily reproducible techniques for studying protein lipidation, the covalent attachment of lipids to proteins. These cutting-edge methods-many never published before in a "hands-on" format-deal with glycosyl phosphatidylinositol (GPI)-containing compounds, protein protein fatty acylation, and protein prenylation. Included are novel techniques for determining the chemical structure of GPI-anchors, for radiolabeling the prenyl groups of protein in eukaryotic cells, a tool for developing inhibitors of the protein farnesyltransferase, and for an exciting lysosomal enzyme that cleaves fatty acyl groups from proteins, the first fatty acylase discovered. Protein Lipidation Protocols offers biochemists, cell and molecular biologists, medicinal chemists, and pharmaceutical researchers state-of-the-art tools for understanding the complex biochemistry of protein lipidation.
E-Book Content
Methods in Molecular Biology TM VOLUME 116 Protein Lipidation Protocols Edited by Michael H. Gelb HUMANA PRESS In Vitro Analysis of GPI Biosynthesis 1 1 In Vitro Analysis of GPI Biosynthesis in Mammalian Cells Victoria L. Stevens 1. Introduction 1.1. Background 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 biosyntheti