Bell J B, Jones M E
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
J Biol Chem. 1991 Jul 5;266(19):12662-7.
Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.
乳清苷5'-单磷酸脱羧酶（ODCase）在被携带编码ODCase的URA3基因的质粒转化的酵母15C细胞中过表达。在半乳糖诱导9小时后，获得了每升细胞培养物中ODCase活性等同于30毫克纯酶的20克细胞。为了去除酵母蛋白酶，需要进行60%-90%硫酸铵分级分离步骤，并添加乙二胺四乙酸（EDTA）作为金属肽酶的抑制剂。该纯化方案得到了无蛋白酶且在4℃下可稳定储存16个月的ODCase。纯酶在50 mM pH 6的磷酸盐缓冲液中的比活性为40单位/毫克，并且可以在-20℃下于20%甘油中储存，2年多仍保留全部活性。该酶在pH 6和25℃下对乳清苷5'-单磷酸的Km为0.7 microM。通过在变性聚丙烯酰胺凝胶上电泳测定，源自质粒的ODCase单体的分子量为29,500。在低蛋白浓度且不存在与催化位点结合的配体的情况下，ODCase通过蔗糖密度梯度沉降为约30 kDa的单体。增加ODCase浓度或添加作为竞争性抑制剂的配体可诱导沉降速率增加。在所研究的最高蛋白浓度下，或在存在50 mM磷酸盐、933 microM底物（乳清苷5'-单磷酸）或产物（尿苷一磷酸，UMP）的情况下，ODCase沉降为一条典型的46 kDa蛋白质条带。在存在50 microM 6-氮杂尿苷5'-单磷酸或2 microM 1-(5'-磷酸-β-D-呋喃核糖基)巴比妥酸（ODCase的竞争性抑制剂）的情况下，会出现沉降为约64 kDa蛋白质的二聚体。这些结果类似于双功能UMP合酶的ODCase结构域的配体诱导的亚基缔合，并支持将酵母ODCase用作其他物种ODCase的模型。
