PURIFICATION OF LYSOSOMAL MEMBRANE-PROTEINS FROM HUMAN PLACENTA

A major portion of the intracellular hydrolytic reactions are confined to lysosomes, whose membrane proteins take care of the proper lumenal milieu and the release of the degradation products. To obtain materia l for structural characterization of the transport proteins, we elabor ated a procedure for isolation of lysosomal membranes and separation o f their proteins in a two-dimensional electrophoresis. In a first step dense lysosomes were isolated from human placenta using a Percoll gra dient. Subsequently, lysosomal membranes were purified by immunoadsorp tion. The procedure yielded mg-amounts of lysosomal membranes. Protein s associated with lysosomal membranes, acid beta-glucosidase, acetyl-c oenzyme A:alpha-glucosaminide N-acetyltransferase, CD63/LIMP I, and h- lamp-2 were enriched approximately 300-fold as compared to the initial homogenate. Separation of the membrane proteins was achieved in a two -dimensional electrophoresis. The procedure is expected to yield mater ial for structural studies on lysosomal membrane proteins with suspect ed defects in lysosomal storage diseases.