Purification and characterization of plasma membranes from Physarum polycephalum amoebae

Abstract

In this study we describe a method for preparing plasma membranes from Physarum polycephalum amoebae. The cells were swollen in hypotonic medium (1 mM ZnCl 2, 10 mM Tris-HCl, pH 8.0) and then broken in a Thomas tissue grinder fitted with a teflon pestle. The plasma membranes were collected by differential centrifugation and purified by centrifugation on a continuous 20–50% sucrose gradient. The membranes sedimented in a single band having a density of 1.16 g/cm 3. They were found by enzymatic assay and by electron microscopy to be free of lysosomes, mitochondria, and nuclei and minimally contaminated by endoplasmic reticulum. The membrane proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis; 10 major and 20 minor bands were seen. Periodic acid Schiff's staining of electrophoretically separated proteins revealed five major and six minor bands containing glycoproteins. Radioiodinated cell surface proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis into six bands with apparent weights greater than 68 000.