Expression of recombinant goldfish glutamic acid decarboxylase 65 and evidence for differential pH and PLP responsiveness compared to the human enzyme

Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
Ravinder K SardanaV L Trudeau

Abstract

Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA) that acts as an important inhibitory neurotransmitter in the vertebrate brain, as well as in the regulation of neuroendocrine function. GAD65 and GAD67 are the two main isoforms that exist in vertebrates. The biochemical properties of recombinant forms of goldfish and human GAD65 were examined. The recombinant goldfish GAD65 (gfGAD65) was expressed at high levels using a maltose binding protein fusion system for biochemical characterization. The human GAD65 (hGAD65) was expressed as a GST fusion and was also purified. The recombinant goldfish GAD65 protein has properties that are different from the human counterpart. In particular, the gfGAD65 is less active at acidic pH compared to hGAD65, which is moderately active over a wider range of acidic and basic pH. Interestingly, however, gfGAD65 is less dependent on a cofactor pyridoxal-5'-L-phosphate (PLP) for activity. In the absence of added PLP, cleaved recombinant gfGAD65 showed approximately 20% of maximal activity whereas hGAD65 showed no detectable activity. The physiological and evolutionary significance of these findings is discussed in light of the conserved function ...Continue Reading

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Citations

Dec 11, 2007·Canadian Journal of Physiology and Pharmacology·R AwadJ T Arnason

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