This chapter focuses on the intracellular iodination of lysosome membrane for the studies of membrane composition and recycling. Lactoperoxidase (LPO)-catalyzed iodination is a well-established method for labeling membrane proteins. The chapter describes a method whereby LPO selectively iodinates intracellular membranes within living macrophages (Mϕ). The LPO is covalently coupled to carboxylated polystyrene latex spheres (LPO-latex). These particles are readily ingested by Mϕ and rapidly established within the phagolysosomal (PL) compartment. When 125I- and H2O2 are added at 4°, the enzyme selectively radiolabels PL polypeptides. The latter can then be compared to radioiodinated plasma membrane (PM) proteins. The labeled PL membrane proteins rapidly return, or recycle, to the cell surface. Therefore, this technique provides a covalent, nonreutilizable label to monitor membrane flow between lysosome and plasma membrane. The coupling procedure is an adaptation of the method of Parikh and Cuatrecasas. In the first step of the coupling procedure, the carboxyl group of CM-latex is activated with a water-soluble carbodiimide so that it is susceptible to nucleophilic attack by N-hydroxysuccinimide (NHS).
ASJC Scopus subject areas
- Molecular Biology