The Membrane Polypeptides of the Vacuolar System: Composition and Recycling

A method has been developed to deliver an iodinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable enzymatically active form. The addition of LPO-Iatex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membranederived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrate both acid phosphatase and lactoperoxidase activities. The exposure of LPO-Iatex containing cells to 125-- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4°C leads to incorporation of label into TCA-precipitable material. The incorporated cell-associated label was present as monoiodotyrosine; negligible amounts were found in lipids. Cell viability remained\u3e 99%. Autoradiography at both the light and EM level revealed that \u3e 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents