Expression of recombinant goldfish glutamic acid decarboxylase 65 and evidence for differential pH and PLP responsiveness compared to the human enzyme.
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 of GAD in two vertebrate species that are separated in evolutionary time by more than 200 million years.
Sardana
Ravinder K
RK
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5.
Awad
Rosalie
R
Arnason
John T
JT
Trudeau
Vance L
VL
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D005982:Glutathione Transferase / Q000378:metabolism; D006054:Goldfish / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D058619:Maltose-Binding Proteins; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014803:Vitamin B Complex / Q000378:metabolism*
D002352:Carrier Proteins; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D014803:Vitamin B Complex; D011732:Pyridoxal Phosphate; D005982:Glutathione Transferase; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2