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

Abstract

A technique is described for isolating nucleoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a sucrose-Tris-MgSO,-KCI-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41,000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the start of induced encystment. No protein changes are observed until the beginning of encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. These nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.Information is now accumulating on the acid-soluble nucleolar proteins from a number of tissues (1, 22~ and on acidic and SDS-soluble nucleolar proteins (7,13,16,33). Extensive electrophoretic analyses of rat liver nucleolar proteins have recently confirmed the great biochemical heterogeneity of these important organelles (22).The nucleolus is known to be the site of synthesis 740

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