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[25] Palmitoylation of G-protein α subunits

1995; Academic Press; Linguagem: Inglês

10.1016/0076-6879(95)50081-2

1557-7988

Maurine E. Linder, Christiane Kleuss, Susanne M. Mumby,

Amino Acid Enzymes and Metabolism

This chapter describes the procedures used to detect protein palmitoylation. The protocols for metabolic radiolabeling of cells and for analysis of radioactive fatty acids incorporated into proteins are also reviewed. Covalent attachment of palmitate to protein occurs usually through a thioester linkage to cysteine. However, the lipid may also be bound to protein through oxyester linkages to serine or threonine. Fatty acylation of proteins through ester bonds is not specific for palmitate. Longer chain saturated fatty acids, such as stearate, are also detected. Modification of proteins by myristate is primarily by cotranslational addition of the fatty acid through an amide linkage to the amino terminus of the protein. Protein palmitoylation contributes to membrane association of a number of proteins including G-protein α subunits, and the neuronal growth cone protein GAP-43. Palmitoylation of proteins is usually assayed by isolating the protein from cells that have been incubated with radioactive palmitic acid. Thioester linkages are hydrolyzed by treatment with base, and fatty acids cleaved from the protein can be analyzed by reversed phase high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).