Purification and Characterization of a High Molecular Weight Histone Deacetylase Complex (HD2) of Maize Embryos

• Published in: Biochemistry (Easton) 1996-12, Vol.35 (49), p.15907-15914
• Main Authors: Brosch, Gerald, Lusser, Alexandra, Goralik-Schramel, Maria, Loidl, Peter
• Format: Article
• Language: English
• Subjects: Blotting, Western; Chromatin - chemistry; Chromatography, Agarose; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Histone Deacetylases - chemistry; Histone Deacetylases - isolation & purification; Isoenzymes - chemistry; Plant Proteins - chemistry; Sequence Analysis; Sequence Homology, Amino Acid; ZEA MAYS; Zea mays - enzymology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi961294x

The dynamic state of core histone acetylation is maintained by histone acetyltransferases and deacetylases. In germinating maize embryos, four nuclear histone deacetylases can be distinguished. From a chromatin fraction prepared at 72 h after start of embryo germination, we have purified the nuclear histone deacetylase HD2 to homogeneity. Using a sequence of chromatographic steps, we achieved the purification of an enzymatically active high molecular weight protein complex with an apparent molecular mass of 400 kDa, as determined by gel filtration chromatography. The purified enzyme was characterized in terms of enzymatic and kinetic properties, and sensitivity to several histone deacetylase inhibitors. In SDS−polyacrylamide gels, HD2 split into three polypeptides of 45, 42, and 39 kDa, suggesting that the native enzyme is a multimer−protein complex. Electrophoresis under nondenaturing conditions in combination with second dimension SDS−gel electrophoresis indicated that all three protein components of the HD2 complex were enzymatically active. Polyclonal antibodies against each of the three polypeptides were raised in rabbits. Each antiserum reacted with all three polypeptides on Western blots, suggesting that p45, p42, and p39 are highly homologous. This homology was confirmed by amino acid sequencing of peptides generated from each of the three HD2 components.