Denner L A, Wei S C, Lin H S, Lin C T, Wu J Y
Proc Natl Acad Sci U S A. 1987 Feb;84(3):668-72. doi: 10.1073/pnas.84.3.668.
Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC 4.1.1.15) was purified from whole rat brain approximately equal to 1300-fold to apparent homogeneity with a specific activity of 2.4 units per mg of protein by a combination of column chromatographies on DEAE-cellulose, hydroxylapatite, and gel filtration, and preparative nondenaturing polyacrylamide gel electrophoresis. The purified preparation contained a single protein band that comigrated with GDCase activity in three diverse analyses: nondenaturing regular (5%) and gradient (3.6-25%) polyacrylamide gel electrophoresis and isoelectric focusing at pH 4-7. The native molecular mass was calculated to be 120 +/- 10 kDa from gradient polyacrylamide gel electrophoresis and 110 +/- 10 kDa from gel filtration. Under the treatment with NaDodSO4 and 2-mercaptoethanol, GDCase dissociated into two subunits of 40 +/- 2 and 80 +/- 4 kDa, as estimated from NaDodSO4 gel electrophoresis. However, only a 40-kDa subunit was detected when GDCase was treated with 4 M urea plus NaDodSO4 and 2-mercaptoethanol, suggesting that the 80-kDa subunit is the dimer of the 40-kDa subunit. In immunoblotting, polyclonal antibodies against GDCase reacted with both 40- and 80-kDa subunits, while monoclonal antibody reacted with only 80-kDa subunits. The isoelectric point of the native enzyme was 5.4. The Km for glutamate was 1.59 X 10(-3) M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at approximately equal to 10% of the rate of glutamate. The pH optimum was fairly broad, with a maximum at approximately equal to 7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and beta-methylene-DL-aspartate.
谷氨酸脱羧酶（GDCase；L-谷氨酸-1-羧基裂解酶，EC 4.1.1.15）通过DEAE-纤维素柱色谱、羟基磷灰石柱色谱、凝胶过滤以及制备性非变性聚丙烯酰胺凝胶电泳等方法从全大鼠脑中纯化得到，纯化倍数约为1300倍，达到表观均一性，比活性为每毫克蛋白质2.4单位。纯化后的制剂在三种不同分析中含有一条与GDCase活性共迁移的单一蛋白带：非变性常规（5%）和梯度（3.6 - 25%）聚丙烯酰胺凝胶电泳以及pH 4 - 7的等电聚焦。根据梯度聚丙烯酰胺凝胶电泳计算，天然分子量为120±10 kDa，根据凝胶过滤计算为110±10 kDa。在十二烷基硫酸钠（NaDodSO4）和2-巯基乙醇处理下，根据NaDodSO4凝胶电泳估计，GDCase解离为两个亚基，分子量分别为40±2 kDa和80±4 kDa。然而，当GDCase用4 M尿素加NaDodSO4和2-巯基乙醇处理时，仅检测到一个40 kDa的亚基，这表明80 kDa的亚基是40 kDa亚基的二聚体。在免疫印迹中，针对GDCase的多克隆抗体与40 kDa和80 kDa亚基均发生反应，而单克隆抗体仅与80 kDa亚基发生反应。天然酶的等电点为5.4。谷氨酸的Km值为1.59×10⁻³ M。除了L-谷氨酸外，半胱氨酸亚磺酸也以约为谷氨酸脱羧速率10%的速度进行脱羧反应。最适pH相当宽，在约7.3时达到最大值。该酶受到羰基捕获剂、巯基试剂、硫醇化合物和β-亚甲基-DL-天冬氨酸的强烈抑制。
