An Improved Method for Production of Recombinant Human Glutamic Acid Decarboxylase 65 for Use in Phytopharmaceutical Assessment

Abstract

The pharmacological activity of neuroactive phytochemicals on recombinant human glutamic acid decarboxylase 65 (hGAD65) was investigated. GAD catalyzes the conversion of glutamic acid to γ-aminobutyric acid (GABA), which acts as an important inhibitory neurotransmitter in the central nervous system (CNS). We describe an improved method in which recombinant hGAD65 was expressed at high levels using a maltose binding protein (MBP) fusion system. The expression and purification process was superior to the commonly used glutathioneS.-transferase (GST) fusion partner. The in vitro. system developed here detected both enzyme inhibition and stimulation, under varying pyridoxal-5′-phosphate (PLP) concentrations. The known GAD inhibitor, 3-mercaptopropionic acid, was tested as a positive control and had an IC₅₀ = 12.3 μ M. Phytochemicals were tested (10 μ g/mL) for their effects on in vitro. hGAD65 activity. Minor inhibition was seen with the ethanol extract of Panax quinquefolius. L. (ginsenosides) (23%), betulinic acid (27%), and valerenic acid (20%). An increase in hGAD65 activity by approximately 20% was observed with bilobalide and asiaticoside. As a result, these small changes in GAD activity may have physiologic implications. The possibility that phytochemicals influence GABAergic neurotransmission in vivo. and the mechanisms by which it may occur is discussed.

Keywords:

• Asiaticoside
• bilobalide
• ginsenosides
• maltose binding protein fusion
• recombinant human GAD65
• valerenic acid

Notes

*Dedicated to Professor John Thor Arnason of the University of Ottawa, Department of Biology, on the occasion of his sixtieth birthday.