Properties and interaction of heterologously expressed glutamate decarboxylase isoenzymes GAD(65kDa) and GAD(67kDa) from human brain with ginkgotoxinand its 5 '-phosphate

Two isoforms of glutamate decarboxylase (GAD(65kDa) and GAD(67kDa)) from hu man brain, which had previously been overexpressed in Escherichia coli as f usion proteins containing a glutathione-S-transferase domain; were purified by affinity chromatography on glutathione Sepharose 4B. Both isoforms were also expressed in Saccharomyces cerevisiae. After modification of a HPLC b ased assay, the enzymes were characterized: with respect to their biochemic al properties. Comparison of kinetic data, pH, and temperature optima as we ll as of the mode of interaction with pyridoxal phosphate as a cofactor rev ealed several significant differences between the two isoenzymes reflecting their somewhat different physiological and molecular features. Investigati on of the influence of 4 ' -O-methylpyridoxine (ginkgotoxin) (1), a neuroto xin occurring in Ginkgo biloba L., on the different isoenzymes, indicates t hat the phosphorylated form of the toxin, 4 ' -O-methylpyridoxine-5 ' -phos phate (2), decreases GAD(65kDa) activity, although in unphysiologically hig h concentrations, whereas GAD(67kDa) activity seems to be hardly affected.