The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum.
A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.
D000469:Alkaline Phosphatase / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003579:Cytochrome Reductases / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005615:Freezing; D007454:Iodides; D007457:Iodine Radioisotopes; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology; D009235:Myxomycetes / Q000032:analysis*; D009249:NADP; D009584:Nitrogen; D009708:Nucleotidases / Q000032:analysis; D013053:Spectrophotometry; D013347:Subcellular Fractions; D013385:Succinate Dehydrogenase / Q000032:analysis
D007454:Iodides; D007457:Iodine Radioisotopes; D009249:NADP; D013385:Succinate Dehydrogenase; D003579:Cytochrome Reductases; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D009584:Nitrogen