Attisano L, Lewis P N
Department of Biochemistry, University of Toronto, Canada.
J Biol Chem. 1990 Mar 5;265(7):3949-55.
Two forms of porcine histone acetyltransferase (types I and II) have been purified to apparent homogeneity from liver nuclei. Both activities are extracted from nuclei by 0.5 M NaCl and display a native Mr of 110,000 as determined by gel filtration. Saline enzyme extracts were subject to ammonium sulfate precipitation and sequential chromatography on Q-Sepharose, Sephacryl S-200, hydroxylapatite, and Mono Q supports. The histone acetyltransferase type I fraction contains three polypeptide chains with apparent Mr values of 105,000, 62,000, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide peptide mapping and immunoblotting suggest that the Mr 62,000 and 45,000 polypeptides are derived by cleavage of the Mr 105,000 polypeptide. Histone acetyltransferase type II contains two different subunits with apparent Mr values of 50,000 and 40,000, respectively. The amino acid composition, heat inactivation profiles, and Michaelis constants with respect to both acetyl coenzyme A and histones were indistinguishable for types I and II. However, affinity-purified polyclonal antibodies to both forms of the enzyme do not cross-react; cyanogen bromide-derived in situ cleavage digest patterns show few similarities; and the turnover number for type I is approximately 15-fold lower than that for type II. We estimate that there is one enzyme molecule for every 500 nucleosomes. The existence of two distinct forms of nuclear histone acetyltransferase in pig liver suggests that they may have separate functions in vivo.
已从猪肝细胞核中纯化出两种形式的猪组蛋白乙酰转移酶（I型和II型），纯度达到表观均一。两种活性均通过0.5M NaCl从细胞核中提取出来，经凝胶过滤测定，其天然分子量为110,000。盐溶液酶提取物先进行硫酸铵沉淀，然后依次在Q-琼脂糖凝胶、Sephacryl S-200、羟基磷灰石和Mono Q支持物上进行层析。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，I型组蛋白乙酰转移酶组分含有三条多肽链，其表观分子量分别为105,000、62,000和45,000。溴化氰肽图谱分析和免疫印迹表明，分子量为62,000和45,000的多肽是由分子量为105,000的多肽裂解而来。II型组蛋白乙酰转移酶含有两个不同的亚基，其表观分子量分别为50,000和40,000。I型和II型在氨基酸组成、热失活曲线以及相对于乙酰辅酶A和组蛋白的米氏常数方面没有差异。然而，针对这两种酶形式的亲和纯化多克隆抗体不发生交叉反应；溴化氰原位裂解消化模式显示几乎没有相似之处；I型的周转数比II型低约15倍。我们估计每500个核小体中有一个酶分子。猪肝中存在两种不同形式的核组蛋白乙酰转移酶，这表明它们在体内可能具有不同的功能。
