Distinct site specificity of two pea histone deacetylase complexes.
We report on the site specificity of two intact pea histone deacetylase complexes. HD1 deacetylates lysines 5 and 16 of H4 in the order K16 > K5, while in the case of H3 the preferred order is K4 >> K18 approximately K9. The specificity of the HD2 complex is markedly different. The preferred residues in H4 are K8 approximately K5 > K16, while in H3 deacetylation, the complex HD2 prefers sites 4 and 18. To obtain these results, we have used a novel procedure based on the SPOT technique, a method to synthesize peptides on membrane supports. Different sets of membranes with sequentially overlapping histone peptides containing acetylated lysines in the sites corresponding to all in vivo acetylatable residues were incubated with the complexes. The acetyl groups removed by the deacetylase activity were then replaced by radioactive acetate by treating the membranes with labeled acetic anhydride. The subsequent counting of the membranes allows the quantification of the acetate removal in the histone deacetylase reaction in a way that circumvents some of the inconveniences of other available procedures.
Clemente
S
S
Department of Biochemistry and Molecular Biology, University of Valencia, E-46100 Burjassot, Valencia, Spain.
Franco
L
L
López-Rodas
G
G
D006655:Histone Deacetylases / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology*; D013379:Substrate Specificity
D006655:Histone Deacetylases