Abstract

Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.

摘要

许多研究集中在调节 γ-氨基丁酸转氨酶 (GABA-T)（一种 GABA 分解代谢酶）的活性，用于治疗神经系统疾病，如癫痫和毒瘾。然而，人类 GABA-T 合成和纯化尚未建立。因此，GABA-T 的生化和药物设计研究主要通过使用猪 GABA-T 甚至细菌 GABA-T 进行。在这里，我们报告了一个优化的协议，用于在人类细胞中过表达 6xHis 标记的人类 GABA-T，然后进行两步蛋白质纯化。然后，我们建立了优化的人类 GABA-T (0.5 U/mg) 活性测定。最后，我们比较了人和细菌 GABA-T 在对两种不可逆 GABA-T 抑制剂（加巴克林和氨己烯酸）的敏感性方面的差异。同二聚体形式的人类 GABA-T 对氨己烯酸的敏感性比多聚体形式的细菌 GABA-T 高 70 倍，表明使用人类 GABA-T 的重要性。总之，我们新开发的协议可以成为开发更有效的人类 GABA-T 调节剂的重要第一步。