露花叶片PEP羧化酶的纯化及某些分子特性

经硫酸铵分部沉淀,DEAE-纤维素(DE52),DEAE-Sephadex A-50,SephacrylS-200和二次羟基磷灰石等柱层析,从露花叶片中分离得到纯化63.9倍、电泳均一的磷酸烯醇式丙酮酸羧化酶。此酶的天然分子量经聚丙烯酰胺梯度凝胶电泳测定为260kD,经SephadexG-200凝胶过滤法测定为240kD。用SDS-聚丙烯酰胺梯度凝胶电泳测得酶的亚基分子量为115kD,表明此酶是个二同聚体。此酶的等电点为PI=5.6。免疫双扩散的结果表明此酶与高梁PEPG的抗原决定簇呈部分同一性。

Purification and Some Molecular Properties of PEP-carboxylase from Mesembryanthemun cordifolium Leaves

The major isozyme of PEP-carboxylase [EC 4.1.1.31] in M. cordifolium leaves has been purified approximately 64 fold with about 30% recovery (Table 1) by means of ammonium sulfate fractionation and column chromatography on DEAE-Cellulose, DEAESephadex A-50, Sephaeryl S-200 (Fig. 1) and hydroxylapatite (Fig. 2). The purified enzyme was shown to be homogeneous by SDS-polyacrylamide gel electrophoresis (Fig. 3). The enzyme had a native molecular weight of 260,000 daltons and 240,000 daltons determined with gradient polyacrylamide gel electrophoresis (Fig. 4) and Sephadex G-200 filtration (Fig. 5), respectively. SDS-gradient polyacrylamide gel electrophoresis showed that this enzyme was a dimer of two identical subunits with molecular weight of 115,000 daltons (Fig. 3). The enzyme had an isoelectric point at 5.6 (Fig. 6) and showed partial identity with photosynthetic PEP-earboxylase of sorghum in immunodiffusion (Fig. 7).