Benidickson Kirsten H, Symonds Kyle F, Snedden Wayne A, Plaxton William C
Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Biol Methods Protoc. 2025 Jun 20;10(1):bpaf050. doi: 10.1093/biomethods/bpaf050. eCollection 2025.
γ-aminobutyrate (GABA) is a non-proteinogenic amino acid produced by glutamate decarboxylase (GAD) that functions as a vital neurotransmitter in animals, and as an important metabolite and signaling molecule in plants and microbes. "GABase" consists of a mixture of recombinant GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSDH) that is widely used for spectrophotometric quantification of glutamate decarboxylase (GAD) activity or GABA levels in tissue extracts. Both can be conveniently monitored at 340 nm owing to the sequential conversion of GABA into succinate by GABA-T and SSDH, and concomitant reduction of NADP into NADPH by SSDH. Currently, these assays rely on commercially available GABase from . However, the excessive cost of commercial GABase prompted us to develop an inexpensive and rapid "DIY" method for producing GABase by cloning, expressing and purifying His-tagged GABA-T and SSDH from . We validated our in-house GABase preparation by comparing GAD activities and GABA levels of the model plant with those obtained using commercial GABase. Both plasmids for expressing His-GABA-T and His-SSDH have been deposited into AddGene (www.addgene.com). Our protocols for producing and using recombinant GABase should be of interest to any researcher who studies eukaryotic or prokaryotic GABA and/or GAD activity.
γ-氨基丁酸（GABA）是一种由谷氨酸脱羧酶（GAD）产生的非蛋白质氨基酸，它在动物体内作为重要的神经递质发挥作用，在植物和微生物中作为重要的代谢物和信号分子。“GABase”由重组GABA转氨酶（GABA-T）和琥珀酸半醛脱氢酶（SSDH）的混合物组成，广泛用于分光光度法定量组织提取物中的谷氨酸脱羧酶（GAD）活性或GABA水平。由于GABA-T和SSDH将GABA依次转化为琥珀酸，以及SSDH将NADP伴随还原为NADPH，两者都可以在340nm处方便地监测。目前，这些测定依赖于市售的GABase。然而，市售GABase的高昂成本促使我们开发一种廉价且快速的“自制”方法，通过从克隆、表达和纯化His标签的GABA-T和SSDH来生产GABase。我们通过比较模式植物的GAD活性和GABA水平与使用市售GABase获得的结果，验证了我们内部制备的GABase。用于表达His-GABA-T和His-SSDH的两种质粒已保藏于AddGene（www.addgene.com）。我们生产和使用重组GABase的方案应该会引起任何研究真核或原核GABA和/或GAD活性的研究人员的兴趣。
