Eberharter A, Lechner T, Goralik-Schramel M, Loidl P
Department of Microbiology, University of Innsbruck, Medical School, Austria.
FEBS Lett. 1996 May 13;386(1):75-81. doi: 10.1016/0014-5793(96)00401-2.
From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.
从玉米胚的可溶性细胞组分中，我们纯化得到了一种细胞质组蛋白乙酰转移酶，其纯度达到了表观均一，该酶符合B型酶的所有标准。通过8个色谱步骤，我们对一种分子量约为90 kDa的具有酶活性的蛋白质进行了6700倍的纯化。在变性条件下，该蛋白质分解为两个组分，在SDS-PAGE中分别以45 kDa和50 kDa的分子量迁移，这表明天然酶是一种异二聚体。对纯化后的酶进行了物理化学性质、动力学性质和底物特异性方面的表征。它对组蛋白H4具有特异性，能在体外将未乙酰化的H4亚类乙酰化为二乙酰化状态。其活性与胚萌发过程中分生细胞内DNA复制的强度一致。我们建立了一种非变性条件下的电泳系统，用于检测凝胶基质中的酶活性；结合二维SDS-PAGE，该方法即使在粗酶制剂中也能明确鉴定组蛋白乙酰转移酶。
