Spink D C, Porter T G, Wu S J, Martin D L
Biochem J. 1985 Nov 1;231(3):695-703. doi: 10.1042/bj2310695.
Pig brain contains three forms of glutamate decarboxylase with pI values of 5.3, 5.5 and 5.8, referred to as the alpha-, beta- and gamma-forms respectively. These forms were purified and kinetically characterized. The major synaptic form of glutamate decarboxylase (the beta-form) migrated as a single band on electrophoresis in sodium dodecyl sulphate/polyacrylamide gels with an apparent Mr of 60 000. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by immunoblotting with an affinity-purified antibody to the enzyme indicated a subunit Mr of 60 000 for the alpha- and gamma-forms as well. An extensive kinetic analysis, aided by an integrated equation that describes the inactivation and re-activation cycle of the enzyme, revealed that the three forms of the enzyme differ markedly in kinetic properties. The Km values for L-glutamate were 0.17, 0.45 and 1.24 mM respectively for the alpha-, beta- and gamma-forms. The Ki for 4-aminobutyrate, the first-order rate constants for inactivation by L-glutamate and 4-aminobutyrate, the rate constant for re-activation of the apoenzyme by pyridoxal 5'-phosphate and the dissociation constant for pyridoxal 5'-phosphate also differed in a similar way among the three forms; the values were in the order alpha-form less than beta-form less than gamma-form.
猪脑含有三种形式的谷氨酸脱羧酶，其等电点分别为5.3、5.5和5.8，分别称为α-、β-和γ-形式。这些形式被纯化并进行了动力学表征。谷氨酸脱羧酶的主要突触形式（β-形式）在十二烷基硫酸钠/聚丙烯酰胺凝胶中电泳时迁移为单一条带，表观相对分子质量为60000。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳后，用针对该酶的亲和纯化抗体进行免疫印迹分析表明，α-和γ-形式的亚基相对分子质量也为60000。借助描述该酶失活和再激活循环的积分方程进行的广泛动力学分析表明，该酶的三种形式在动力学性质上有显著差异。α-、β-和γ-形式的L-谷氨酸的Km值分别为0.17、0.45和1.24 mM。4-氨基丁酸的Ki、L-谷氨酸和4-氨基丁酸失活的一级速率常数、吡哆醛5'-磷酸使脱辅酶再激活的速率常数以及吡哆醛5'-磷酸的解离常数在三种形式中也以类似方式存在差异；其值的顺序为α-形式＜β-形式＜γ-形式。
