DECREASED HETEROGENEITY OF CS HISTONE VARIANTS AFTER HYDROLYSIS OF THE ADP-RIBOSE MOIETY

Sea urchin CS histone variants are electrophoretically heterogeneous w hen analyzed in two dimensional polyacrylamide gels (2D-PAGE). Previou s results suggested that this heterogeneity is due to the poly (ADP-ri bosylation) of these proteins. Consequently, native CS histone variant s were subjected to different treatments to remove the ADP-ribose moie ty. The incubation in 1 M hydroxylamine was not effective in eliminati ng the polymers of ADP-ribose from CS variants, and the treatment with sodium hydroxide was deleterious to the proteins. In contrast, the AD P-ribose moiety was successfully removed from the CS variants by incub ation with phosphodiesterase (PDE). To eliminate contamination of CS h istone variants with PDE extract, the enzyme was covalently bound to S epharose 4B prior to its utilization. Treatment of native CS histone v ariants with this immobilized phosphodiesterase removed around 85% of the total ADP-ribose moiety from these proteins. After S-PDE treatment the complex electrophoretic pattern of CS histone variants in 2-D PAG E decreases to five major fractions. From these results we conclude th at the electrophoretic heterogeneity of native CS histone variants is mainly due to the extent to which five main CS histone variants are po ly(ADP)-ribosylated). (C) 1996 Wiley-Liss, Inc.