Protein Lipidation Protocols

About this book

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.

Contents

Selection of Mammalian Cell Mutants in GPI Biosynthesis In Vitro Analysis of GPI Biosynthesis in Mammalian Cells . Analysis of the cell surface distribution of GPI-anchored proteins Imaging Fluorescence Resonance Energy Transfer as a Probe of the Membrane Organization and Molecular Associations of GPI-Anchored Proteins Purification of Caveolae-Derived Membrane Microdomains Containing Lipid-Anchored Signaling Molecules Such as GPI-Anchored Proteins H-Ras Src-Family Tyrosine Linases eNOS and G-Protein a b and g Subunits Analysis of Lipids in Caveolae Analysis of the Carbohydrate Components of Glycosylphosphatidylinositol Structures Using Fluorescent Labeling Analysis of the Lipid Moiety of GPI Anchor in the Yeast Saccharomyces cerevisiae Fulvio Reggiori Rapid Identification of Cysteine-Linked Isoprenyl Groups By Metabolic Labeling with [3H]Farnesol and [3H]Geranylgeraniol Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into Mammalian Cell Proteins: A Useful Tool for Studying Protein Prenylation Reconstitution of Yeast Farnesyltransferase from Individually Purified Subunits Probing the Role of H-Ras Lipidation for Signaling Functions in Xenopus laevis oocytes Fluorescence Measurement of Lipid-Binding Affinity and Interbilayer Transfer of Bimane-Labeled Lipidated Peptides Determination of the Kinetics of Intervesicle Transfer and Transbilayer Diffusion of Bimane-Labeled Lipidated Peptides Preparation and Assay of Myristoyl-CoA:Protein N-Myristoyltransferase Fatty Acid Analysis of Protein-Derived Lipid Thioesters Isolated from Palmitoyl-Protein Thioesterase-Deficient Cells Metabolic Labeling of Protein-Derived Lipid Thioesters in Palmitoyl-Protein Thioesterase-Deficient Cells

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