Protein Lipidation Protocols

ISBN-13: 9781489943309 / Angielski / Miękka / 2013 / 240 str.

Michael H. Gelb

Opis

It is hard to think of a protein in eukaryotic cells that does not undergo some type of posttranslational modification. The covalent attachment of l- ids to proteins, protein lipidation, occurs for a few thousand proteins. Several functions for protein lipidation are known. Protein lipids may target proteins to specific cellular membranes, they may serve as molecular switches that allow cytosol-to-membrane transfer, they may direct protein-protein compl- ation, and they may stabilize protein structure. In cases such as the fatty a- lation of intracellular loops of transmembrane proteins, the funtions of the protein lipidations are not known. This volume-Protein Lipidation Protocols-provides detailed meth- ologies for the study of these processes. Since this is a rapidly growing field, many new experimental techniques have been developing over the past few years. All of the experimental techniques described in this volume have emerged during this time. The editor has made a special effort to include only those techniques that have not been previously described in a "hands-on" f- mat.

Spis treści

In Vitro Analysis of GPI Biosynthesis in Mammalian Cells, Victoria L. Stevens. Selection of Mammalian Cell Mutants in GPI Biosynthesis, Victoria L. Stevens. Analysis of the Cell-Surface Distribution of GPI-Anchored Proteins, Satyajit Mayor. Imaging Fluorescence Resonance Energy Transfer as Probe of Membrane Organization and Molecular Associations of GPI-Anchored Proteins, Anne K. Kenworthy and Michael Edidin. 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, Michael P. Lisanti, Massimo Sargiacomo, and Philipp E. Scherer. Analysis of Lipids in Caveolae, Jun Liu and Jan E. Schnitzer. Analysis of the Carbohydrate Components of Glycosylphosphatidylinositol Structures Using Fluorescent Labeling, Nicole Zitzmann and Michael A. J. Ferguson. Analysis of the Lipid Moiety of GPI Anchor in the Yeast Saccharomyces cerevisiae, Fulvio Reggiori, Elisabeth Canivenc-Gansel, and Andreas Conzelmann. Rapid Identification of Cysteine-Linked Isoprenyl Groups By Metabolic Labeling with [3H]Farnesol and [3H]Geranylgeraniol, Douglas A. Andres, Dean C. Crick, Brian S. Finlin, and Charles J. Waechter. Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into Mammalian Cell Proteins: A Useful Tool for Studying Protein Prenylation, Alberto Corsini, Christopher C. Farnsworth, Paul McGeady, Michael H. Gelb, and John A. Glomset. Reconstitution of Yeast Farnesyltransferase from Individually Purified Subunits, Jun Urano and Fuyuhiko Tamanoi. Probing the Role of H-Ras Lipidation for Signaling Functions in Xenopus laevis Oocytes, Thomas Dudler and Michael H. Gelb. Fluorescence Measurement of Lipid-Binding Affinity and Interbilayer Transfer of Bimane-Labeled Lipidated Peptides, John R. Silvius. Determination of the Kinetics of Intervesicle Transfer and Transbilayer Diffusion of Bimane-Labeled Lipidated Peptides, John R. Silvius. Preparation and Assay of Myristoyl-CoA: Protein N-Myristoyltransferase, Rajala V. S. Raju and Rajendra K. Sharma. Metabolic Labeling of Protein-Derived Lipid Thioesters in Palmitoyl-Protein Thioesterase-Deficient Cells, Sandra L. Hofmann and Jui-Yun Lu. Fatty Acid Analysis of Protein-Derived Lipid Thioesters Isolated from Palmitoyl-Protein Thioesterase-Deficient Cells, Sandra L. Hofmann and Linda A. Verkruyse. Index.

Blurb

In Protein Lipidation Protocols, Michael Gelb brings together a 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 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, as well as catalyzing the development of many important new biopharmaceuticals, including anticancer drugs.