Synthesis and Use of Iodo-Fatty Acid Analogs

Abstract

This chapter describes the synthesis of the iodo-fatty acid analogs and their uses in detecting acylated proteins in vitro in whole-cell lysates and in isolated mitochondria. Identification of fatty acylated proteins relies on monitoring the incorporation of 3H- or 14C-labeled fatty acids. Radiolabeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and label incorporation is detected by fluorography. A series of [125I]iodo-fatty acids containing a molecule of iodine-125 at the ω carbon is synthesized. The formal replacement of iodine for a methyl group can produce a bioisostere, which is accepted as a substrate by the cellular acylation machinery. Use of radioiodinated fatty acids is advantageous, as it reduces the exposure times required to obtain a signal and allows the use of phosphorimager technology. The high-performance liquid chromatography (HPLC) column is used in purifying the final labeled long-chain fatty acid shielded both during and after use. The two most commonly used sources for in vitro translation of eukaryotic cell proteins—rabbit reticulocyte lysates and wheat germ lysates—contain the enzymatic machinery necessary for protein N-myristoylation. © 1995 Elsevier Inc.