Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos
Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos
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DOI：
10.1021/bi961294x
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发表时间：
1996-12-10
期刊：
影响因子：
2.9
通讯作者：
Loidl, P
中科院分区：
文献类型：
--
作者：
Brosch, G;Lusser, A;Loidl, P
核心组蛋白乙酰化的动态由组蛋白乙酰转移酶和脱乙酰酶维持。在萌发的玉米胚胎中，可以区分出四种核组蛋白脱乙酰酶。从胚胎萌发后72小时制备的染色质组分中，我们纯化了核组蛋白脱乙酰基酶HD2。通过一系列的层析步骤，我们实现了一种具有酶活性的高分子量蛋白质复合体的纯化，凝胶过滤层析测定其表观分子质量为400 kDa。对纯化的酶进行了酶学性质和动力学性质的研究，并对几种组蛋白去乙酰化酶抑制剂进行了敏感性测定。在SDS-聚丙烯酰胺凝胶中，HD2裂解成45、42和39 kDa的三个多肽，表明该天然酶是一个多聚体-蛋白质复合体。非变性条件下的电泳法与双向凝胶电泳法相结合，表明HD2复合体的三种蛋白质组分均具有一定的酶活性。在兔体内分别制备了三种多肽的多克隆抗体。每一抗血清均与所有三种多肽发生免疫印迹反应，提示P45、P42和P39具有高度同源性。通过对三个HD2组分中的每一个产生的多肽进行氨基酸序列测定，证实了这种同源性。
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.