Plasma membranes from Physarum polycephalum Plasmodia: purification, characterization, and comparison with amoebae plasma membranes

Two methods are described for preparing plasma membranes from Physarum polycephalum plasmodia. In the first method Plasmodia were broken in homogenizing medium (250 mM sucrose, 1 mM ZnCl2, 10 mM Tris (pH 8.0)), and the nuclei were removed by low speed centrifugation. A crude membrane fraction was collected and the membranes were purified by centrifugation in a linear 30–65% (w/w) sucrose gradient. The membranes sedimented in a single band at a density of 1.26 g/cm3. In the second method the plasmodia were broken in the same homogenizing medium and a crude membrane fraction was prepared by differential centrifugation. The membranes were purified by the Dextran – polyethylene glycol aqueous two-phase system. Membranes from both preparations were found by enzymatic assay and by electron microscopy to be virtually free of contaminating cell organelles. Analyses by one- and two-dimensional polyacrylamide gel electrophoresis showed a great similarity in the proteins from two-phase and sucrose gradient prepared membranes, indicating that either technique can be used to prepare membranes for protein studies. An analysis by two-dimensional gel electrophoresis of plasma membrane proteins from amoebae and plasmodia showed differences in some of the major proteins, indicating that changes occur in plasma membrane proteins during differentiation.