Imschenetzky M, Morín V, Carvajal N, Montecino M, Puchi M
Department of Molecular Biology, Universidad de Concepción, Chile.
J Cell Biochem. 1996 Apr;61(1):109-17. doi: 10.1002/jcb.12.
Sea urchin CS histone variants are electrophoretically heterogeneous when analyzed in two dimensional polyacrylamide gels (2D-PAGE). Previous results suggested that this heterogeneity is due to the poly (ADP-ribosylation) of these proteins. Consequently, native CS histone variants were subjected to different treatments to remove the ADP-ribose moiety. The incubation in 1 M hydroxylamine was not effective in eliminating the polymers of ADP-ribose from CS variants, and the treatment with sodium hydroxide was deleterious to the proteins. In contrast, the ADP-ribose moiety was successfully removed from the CS variants by incubation with phosphodiesterase (PDE). To eliminate contamination of CS histone variants with PDE extract, the enzyme was covalently bound to Sepharose 4B prior to its utilization. Treatment of native CS histone variants 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 PAGE decreases to five major fractions. From these results we conclude that the electrophoretic heterogeneity of native CS histone variants is mainly due to the extent to which five main CS histone variants are poly(ADP)-ribosylated).
海胆的CS组蛋白变体在二维聚丙烯酰胺凝胶（2D-PAGE）分析时具有电泳异质性。先前的结果表明，这种异质性是由于这些蛋白质的多聚（ADP-核糖基化）所致。因此，对天然的CS组蛋白变体进行了不同处理以去除ADP-核糖部分。在1 M羟胺中孵育对于从CS变体中消除ADP-核糖聚合物无效，而用氢氧化钠处理对蛋白质有害。相比之下，通过与磷酸二酯酶（PDE）孵育，成功地从CS变体中去除了ADP-核糖部分。为了消除CS组蛋白变体被PDE提取物污染，在使用该酶之前将其共价结合到琼脂糖4B上。用这种固定化磷酸二酯酶处理天然CS组蛋白变体可从这些蛋白质中去除约85%的总ADP-核糖部分。经过S-PDE处理后，2-D PAGE中CS组蛋白变体复杂的电泳图谱减少到五个主要部分。从这些结果我们得出结论，天然CS组蛋白变体的电泳异质性主要是由于五种主要CS组蛋白变体的多聚（ADP）核糖基化程度不同所致。
