Glutamate decarboxylase solubilized from the rat cerebral cortex by two different concentrations of Triton X-100: effects of glutamate analogues and analysis by SDS-PAGE/western blotting using GAD6 and K2 antibodies.
Analysis of two preparations (containing 0.1% and 0.5% Triton X-100) of glutamate decarboxylase (GAD) by Western blotting using GAD6 and K2 antibodies specifically recognizing two GAD isoenzymes, GAD65 and GAD67, respectively, indicated that the higher concentration of Triton X-100 at best only moderately favoured solubilization of GAD67. Several glutamate analogues were found to be either equally potent or equally inactive as inhibitors of glutamate decarboxylase activities in the two preparations. Among typical ligands for glutamate receptors and transporters, only quinolinic and L-cysteine sulphinic acids were weak inhibitors of GAD. Kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), 3-((RS)-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), L-threo-3-hydroxy-aspartate, L-trans-pyrrolidine-2,4-dicarboxylate, dihydrokainate, kynurenic acid and N-methyl-D-aspartate were inactive. Even though the activity of glutamate decarboxylase in homogenates of rat cerebral cortex is higher at 0.5% than at 0.1% Triton X-100, structural requirements of the enzyme active site appear to be independent of Triton X-100 concentration. Furthermore, since the less soluble component of the enzyme activity contains about the same ratio of GAD65 to GAD67 as the more soluble one, it does not seem that the fractionation with Triton X-100 can be easily used to separate the two isoenzymes from each other.
Johanson
S O
SO
Department of Anatomy and Histology, University of Sydney, NSW, Australia.
Li
Y
Y
Balcar
V J
VJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000032:analysis*; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D017830:Octoxynol; D051381:Rats; D012995:Solubility
D000911:Antibodies, Monoclonal; D018698:Glutamic Acid; D017830:Octoxynol; D005968:Glutamate Decarboxylase