Isolation and electrophoretic analysis of nucleoli, phenol-soluble nuclear proteins, and outer cyst walls from Acanthamoeba castellanii during encystation initiation.

摘要: A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the F. J. Chlapowski and R. N. Band (1971) are sonicated in surcrose-Tris-MgSO4-KC1-Triton X-100 buffer centrifuged on linear sucrose gradient extending 1.3 M to 1.5 with 2.6 cushion, at 41000 rpm 90 min. The only apparent contaminants nucleolar preparation outer cyst walls. procedure isolation chemically pure walls, comparison proteins reveals that walls represent negligible contaminants. ultrastructure these nucleoli examined transmission electron microscopy found be identical whole cells, fixed an manner. 50 separated SDS gel electrophoresis have been throughout growth cycle into strat induced encystment, which time 10 protein bands disappear, 11 observed decrease, 8 seen increase concentration. Phenol-soluble extracted nucleolus correspond 29 proteins, 17 corresponding change onset encystment. Thes also compared those rat liver electrophoresis, resulting observation extremely few homologies exist between two. Numerous quantitative qualitative changes pattern phenol-soluble nuclear during early late log phase stationary were observed.