Brosch G, Lusser A, Goralik-Schramel M, Loidl P
Department of Microbiology, University of Innsbruck, Medical School, Austria.
Biochemistry. 1996 Dec 10;35(49):15907-14. 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.
核心组蛋白乙酰化的动态状态由组蛋白乙酰转移酶和去乙酰化酶维持。在萌发的玉米胚中，可以区分出四种核组蛋白去乙酰化酶。从胚萌发开始72小时后制备的染色质组分中，我们已将核组蛋白去乙酰化酶HD2纯化至同质。通过一系列色谱步骤，我们实现了对一种具有酶活性的高分子量蛋白质复合物的纯化，经凝胶过滤色谱测定其表观分子量为400 kDa。对纯化的酶进行了酶学和动力学性质以及对几种组蛋白去乙酰化酶抑制剂敏感性的表征。在SDS-聚丙烯酰胺凝胶中，HD2裂解为45、42和39 kDa的三种多肽，表明天然酶是一种多聚体蛋白质复合物。非变性条件下的电泳结合二维SDS-凝胶电泳表明，HD2复合物的所有三种蛋白质组分都具有酶活性。针对这三种多肽中的每一种制备了兔多克隆抗体。每种抗血清在Western印迹上都与所有三种多肽发生反应，表明p45、p42和p39高度同源。通过对由三种HD2组分中的每一种产生的肽段进行氨基酸测序，证实了这种同源性。
