Distinct site specificity of two pea histone deacetylase complexes

We report on the site specificity of two intact pea historic deacetylase co mplexes. HDI deacetylates lysines 5 and 16 of H4 in the order K16 > K5. whi le in the case of H3 the preferred order is K4 much greater than K18 approx imate to K9. The specificity of the HD2 complex is markedly different. The preferred residues in H4 are K8 approximate to K5 > K16, while in H3 deacet ylation, the complex HD2 prefers sites 4 and 18. To obtain these results, w e have used a novel procedure based on the SPOT technique, a method to synt hesize peptides on membrane supports. Different sets of membranes with sequ entially overlapping historic peptides containing acetylated lysines in the sites corresponding to all in vivo acetylatable residues were incubated wi th the complexes. The acetyl groups removed by the deacetylase activity wer e then replaced by radioactive acetate by treating the membranes with label ed acetic anhydride. The subsequent counting of the membranes allows the qu antification of the acetate removal in the histone deacetylase reaction in a way that circumvents some of the inconveniences of other available proced ures.