RADIOMETRIC MICRO-ASSAY FOR GLUTAMIC-ACID DECARBOXYLASE

A simple method for purifying l-[3H]glutamic acid and incubation conditions suitable for estimating l-glutamic acid decarboxylase activity are described. Routine and recycled cation-exchange procedures for separating γ-aminobutyric acid from l-glutamate are outlined and compared. Blanks using the routine cation-exchange method average approximately 0.05% of the radioactivity in the substrate; product recovery is approximately 89%. Recycling increases the sensitivity of the cationexchange method by 6-7-fold. l-Glutamate decarboxylase activity can be measured reliably in samples of embryonic neural tissue having wet-weights of approximately 1 μg using the routine cation-exchange method. The relationship between substrate purity and the sensitivity of the cation-exchange procedures is assessed. Radiochemical purity is the critical determinant of sensitivity for the recycled method. The cation-exchange method is compared with the anion-exchange and CO2-trapping methods. The cation-exchange method is approximately 2-3 orders of magnitude more sensitive than the CO2-trapping method and 1-2 orders of magnitude more sensitive than the anion-exchange method. The cationexchange method is also more specific than either of the other 2 methods. l-Glutamate decarboxylase activity has been detected in the lumbar spinal cord of the chick embryo at Day 2 1 2 (Stage 14) using the cation-exchange method. This is 5-6 days earlier than l-glutamate decarboxylase activity has been detected in embryonic neural tissue by previous investigators. l-Glutamate decarboxylase is present in the lumbar spinal cord at least as early as the birth of the first lumbar spinal cord neurons and at least 1-2 days before the initiation of synaptogenesis. © 1979.