Characterization of a strain-specific inhibitor of cell division from Amoeba cytoplasm

The microinjection of cytoplasm taken from one strain of large free-living amoeba into another strain is followed by an incompatibility phenomenon, the inhibition of division amongst the recipient cells. The post-microsomal supernatant fraction from Amoeba discoides (T1D13) injected into A. proteus (T1P) inhibited division in 90% of the injected cells. Further centrifugation of this fraction yielded a pellet which when resuspended and injected, inhibited division in over 95% (and sometimes 100%) of the cells. No inhibitory activity remained in the supernatant after the removal of this pellet. Treatment with 10 micrograms/ml trypsin destroyed the activity of this pellet, while 25 micrograms/ml ribonuclease reduced the inhibitory activity by approximately 40%. Passage of the resuspended post-microsomal pellet through Sephadex G-200 gave one main peak of material which eluted in the void volume. Concentration of this material by either dialysis or lyophilization followed by microinjection into A. proteus showed that this void volume peak contained the inhibitory material, although the most active preparations did not give more than 66% inhibition of division. After elution from Sephadex, the void volume material was analysed by electrophoresis under non-denaturing and denaturing conditions, and by isoelectric focusing. One problem was the loss of inhibitory activity after keeping the pellet at 4 degrees C for 4-5 days, which made further analysis by microinjection difficult. Preliminary experiments using a post-microsomal pellet prepared from Dawson's A. proteus (DP) which inhibited division in A. proteus (T1P) gave a similar profile after Sephadex chromatography and gel electrophoresis.