'Ghosts' formation and their isolation from the cellular slime mold Dictyostelium discoideum.

Conditions for isolating ghosts from the cellular slime mold Dictyostelium discoideum are described. The cells were washed with a 20 mM KCl, 2.5 mM MgCl₂ solution and homogenized vigorously in 15 mM lactate buffer, pH 4.8, using a tight-fitting Dounce homogenizer. The resultant spherical ghosts were purified by the dextran-polyethylene glycol aqueous two-phase system described by Brunette &; Till [1], The proportion of ghosts which are finally purified by 3rd partition in the aqueous two-phase system is 5.6% of those present in the homogenate. As shown by phase-contrast and scanning electron microscopy, the plasma membrane fractions are almost completely uncontaminated by other identifiable subcellular components. On the basis of enzyme assays the ghosts isolated showed a 9- to 11-fold enrichment of alkaline phosphatase relative to the homogenate. They are free of succinic dehydrogenase, glucose 6-phosphatase but do contain some acid phosphatase and N-acetylglucosaminidase activity. Further purification using a sucrose-density gradient removes the residual lysosomal enzyme activities.