A method for isolation of the plasma membrane of the myxamoebae and swarm cells of the myxomycete Didymium iridis using an aqueous two-phase polymer system

Abstract

A method for the isolation of the plasma membranes of the acellular slime mold, "Didymium iridis" was developed using a modification of the dextran-polyethylene glycol aqueous two-phase polymer system. It was found to be superior to the widely accepted technique of density gradient centrifugation, as applied to this particular cell system. A number of chemical and enzymatic assays performed on the membrane and other cell fractions are discussed. These were used a method for positive identification of the plasma membrane and assessment of its purity. The Feulgen reaction was used as an assay for nuclear contamination. Cytochrome oxidase, a mitochondrial marker, and acid phosphatase, a lysosomal marker, were used to check for contamination of the membrane fraction by those two sub-cellular organelles. The membrane preparation was also assayed for the presence of 5 -nucleotidase, an important and characteristic enzyme, present in most plasma membranes and widely used as a marker in its isolation and identification. All steps of the isolation procedure were monitored by phasecontrast and light microscopy.

Results of the chemical and enzymatic assays indicate that plasma membranes are recovered with high yield and purity using a modified two-phase polymer technique. The method is not only rapid, but also can be performed using low-speed centrifugation equipment.