Properties and interaction of heterologously expressed glutamate decarboxylase isoenzymes GAD(65kDa) and GAD(67kDa) from human brain with ginkgotoxin and its 5'-phosphate.
Two isoforms of glutamate decarboxylase (GAD(65kDa) and GAD(67kDa)) from human brain, which had previously been overexpressed in Escherichia coli as fusion 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 based assay, the enzymes were characterized with respect to their biochemical properties. Comparison of kinetic data, pH, and temperature optima as well as of the mode of interaction with pyridoxal phosphate as a cofactor revealed several significant differences between the two isoenzymes reflecting their somewhat different physiological and molecular features. Investigation of the influence of 4'-O-methylpyridoxine (ginkgotoxin) (1), a neurotoxin occurring in Ginkgo biloba L., on the different isoenzymes, indicates that the phosphorylated form of the toxin, 4'-O-methylpyridoxine-5'-phosphate (2), decreases GAD(65kDa) activity, although in unphysiologically high concentrations, whereas GAD(67kDa) activity seems to be hardly affected.
Buss
K
K
Institut für Pharmazeutische Biologie, Universität Bonn, Nussallee 6, 53115 Bonn, Germany.
Drewke
C
C
Lohmann
S
S
Piwonska
A
A
Leistner
E
E
D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D011736:Pyridoxine / Q000031:analogs & derivatives* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013696:Temperature
C406624:4'-O-methylpyridoxine; C435936:4'-O-methylpyridoxine-5'-phosphate; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2; D011736:Pyridoxine