Post-translational Modification of Proteins by Lipids: A Laboratory Manual

The growing interest in recent years in the anchoring to membranes of proteins by post- translational modification is documented by the large number of publications which ap- peared in this field. In September 1987, scientists from 10 countries from all over the world met in the resort village of Les Diablerets, Switzerland, to discuss the most recent advances made in this field. The sessions were devoted to the anchoring of membrane proteins by cova- lent attachment of fatty acids and of glycophospholipids. The workshop brought together many scientists working on vastly different proteins such as the variant surface glycoprotein of Trypanosomes and antigens of the mammalian cells. The subject of the workshop unified many scientists who had not met before and thus greatly stimulated interdisciplinary work. In addition to the lectures, each participant was provided with a collection of Methods currently in use in the study of membrane proteins anchored by post-translational modifica- tion. An updated version of this collection is now presented as a Laboratory Manual, and the techniques described therein will give researchers easy and practical access to the investiga- tion of post-translationally modified proteins. The publication of the present book by Springer follows an established tradition of previously published manuals on the handling of membrane proteins. Our thanks go to the authors who made the essential contribution in writing and adapting the experimental protocols, to Mrs. R.

Table of Contents:
Identification of Glycosyl-Phosphatidylinositol Membrane Anchors by Fatty Acid Labeling.- [3H]Myristate-Labeled Variant Surface Glycoprotein from Trypanosoma brucei: Preparation and Use in the Assay of Glycan-Phosphatidylinositol-Specific Lipases.- Identification of the Glycolipid Anchor of Alkaline Phosphatase by Metabolic Labeling.- Reductive Radiomethylation and Amino Acid Analysis. A Sensitive Procedure to Identify Amine Components in Glycolipid Anchors of Membrane Proteins.- Analytical and Preparative Phase Separation of Glycolipid-Anchored Membrane Proteins in Triton X-114 Solution.- Assay and Purification of Pi-Specific Phospholipase C from Bacillus cereus Using Commercially Available Phospholipase C.- Use of Phenylsepharose to Discriminate Between Hydrophilic and Hydrophobic Forms of Decay Accelerating Factor.- PI-PLC Assay on Nitrocellulose Filter Immobilized Proteins.- Deacylation and Reacylation of a Pure Enzyme to Demonstrate Covalent Glycerolipid.- Incorporation of Rat Brain Thy-1 Antigen into Mouse T Lymphocytes.- Identification and Characterization of Fatty Acid-Acylated Proteins in Cultured Cells by Radiolabeling.- Chemical Analysis of Fatty Acids Covalently Attached to Proteins.- Identification and Characterization of Proteins Undergoing Reversible Fatty Acid Acylation.- Acylation of Proteins in Isolated Mitochondria.- Plant Protein Acylation: Identification of the Modified Proteins and Analysis of the Bound Fatty Acid Ligand.- Identification and Characterization of Lipid-Modified Membrane Proteins in Bacteria.- Detection of Palmitoylating Activity with Exogenous Viral Acceptor Proteins.- Lipid-Mediated Protein Glycosylation: Assembly of Lipid-Linked Oligosaccharides and Post-Translational Oligosaccharide Trimming.- Immunochemical Characterization of the Ia Associated Invariant Chain.- Acylation and Activation of Venom Phospholipase A2 Enzymes by Acyl Imidazolides.- Isolation and Characterization of Bovine Erythrocyte Acetylcholinesterase Solubilized by PI-PLC.- Characterization of Amphiphilic Forms of Cholinesterases by their Interactions with Non-Denaturing Detergents in Centrifugation and Charge-Shift Electrophoresis.