The chemical labeling of glutamate decarboxylase in vivo.

Abstract

Mouse brain glutamate decarboxylase(s) was specifically titrated in vivo and in crude brain homogenates by a combination of gabaculine and [alpha-3H]acetylenic gamma-aminobutyric acid. This specific titration is based on the differential spectra of action of these two mechanism-based enzyme inactivators. The specificity of the titration in vitro was demonstrated by showing that the time course of radioactivity incorporation exactly paralleled the time course for glutamate of decarboxylase inactivation. Furthermore, pretreatment of the crude homogenate with aminooxyacetic acid and alpha-methyl-trans-3-dehydroglutamate, two inactivators of glutamate decarboxylase which function by entirely different mechanisms, decreased count incorporation ([alpha-3H]acetylenic gamma-aminobutyric acid) to the same extent as the activity was decreased. Injection of [alpha-3H]acetylenic gamma-aminobutyric acid intraperitoneally after gabaculine injection led to incorporation of 0.46 nmol of inactivator/mouse brain, when approximatley 70% of the enzyme was inactivated. This means that there is approximately 0.66 nmol of glutamate decarboxylase/0.5 g of mouse brain, assuming the stoichiometry of inactivator bound to enzyme is one. This value is similar to the one (0.646 nmol) obtained from a calculation based on the enzyme purification data (Wu, J.-Y. (1974) in gamma-Aminobutyric Acid in Nervous System Function (Roberts, E., Chase, E. N., and Tower, D. B., eds) pp. 7-55, Raven Press, New York).