title: Biochemical methods for analysis of histone deacetylases.
abstract: Specific lysine residues in the N-terminal extensions of core histones can be posttranslationally modified by acetylation of the epsilon-amino group. The dynamic equilibrium of core histone acetylation is established and maintained by histone acetyltransferases and deacetylases. Both enzymes exist as multiple enzyme forms. Histone acetyltransferases and deacetylases have recently been identified as transcriptional regulators as well as nucleolar phosphoproteins, and have therefore attracted considerable research interest. Analysis of the functional significance of histone deacetylases for nuclear processes in certain cases demands the separation and biochemical analysis of different members of the histone deacetylase families. We have characterized three different histones deacetylases in maize embryos and subsequently purified these enzymes to homogeneity. Here we describe methods for extraction, enzymatic assay, chromatographic and electrophoretic separation, and purification of deacetylases. A novel one-step procedure for large-scale preparation of individual histones and their acetylated isoforms for the analysis of substrate and site specificity of the enzymes is presented.
authors.affiliation: Department of Microbiology, Medical School, University of Innsbruck, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Kölle
authors.forename: G
authors.initials: G
authors.lastname: Brosch
authors.forename: T
authors.initials: T
authors.lastname: Lechner
authors.forename: A
authors.initials: A
authors.lastname: Lusser
authors.forename: P
authors.initials: P
authors.lastname: Loidl
mesh_terms: D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006655:Histone Deacetylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism; D007525:Isoelectric Focusing; D013379:Substrate Specificity
chemical_list: D006657:Histones; D006655:Histone Deacetylases