Rodríguez-Berrocal F J, Páez de la Cadena M, Cabezas J A, Pérez-González N
Department of Biochemistry, Faculty of Biology, University of Salamanca, Spain.
J Biochem. 1988 Jul;104(1):66-71. doi: 10.1093/oxfordjournals.jbchem.a122425.
beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.
β-D-半乳糖苷酶已从兔脾脏中纯化至表观均一。纯化步骤包括硫酸铵沉淀、DEAE-纤维素、伴刀豆球蛋白A-琼脂糖、葡聚糖G-200和琼脂糖4B-(ε-氨基己酰基)-2-脱氧-β-D-葡糖胺亲和层析。在DEAE-纤维素步骤中，β-D-半乳糖苷酶被分离成两种分子形式，分别命名为I和II，它们具有相似的最适pH、Km、底物特异性以及对底物类似物和其他物质的敏感性。形式I纯化了1800倍，产率约为总活性的2%。这种形式对热不稳定，其最适酸性pH为4.0，等电点为6.7，分子量为75,000道尔顿。形式II的最适pH为3.6，有三个不同的pI值(5.3、5.7和6.7)，其相对比例可通过神经氨酸酶处理进行改变。形式II在pH 4.0时似乎是约600,000道尔顿的多聚体形式(IIA)，在中性pH值下可逆地解离为表观分子量为120,000的寡聚体形式(IIB)。IIA和IIB均分别纯化，且均显示酸性最适pH和异质pI(4.6至7.2)。IIA向IIB的解离可自发产生，但洗脱缓冲液中存在尿素时会增加，这表明两者都是共同亚基的聚集体。
