Purification and Characterization of Glutamate Decarboxylase from Mouse Brain

Abstract

Abstract Glutamate decarboxylase catalyzes the formation of γ-aminobutyric acid, an important inhibitory transmitter in invertebrate and vertebrate nervous systems. Approximately 700-fold purification of the enzyme from mouse brain has been achieved by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel, and DEAE-Sephadex chromatography. The most highly purified preparation appeared to be monodisperse on high speed sedimentation equilibrium analysis and was found to have a partial specific volume, v, of 0.732 and a molecular weight of 85,000 ± 2,000. A single major protein band coincident with the enzyme activity was found with polyacrylamide gel. In addition, a faint band also was present. In tests with 20 naturally occurring amino acids and α-ketoglutarate the enzyme showed α-decarboxylation only with glutamate to a great extent; but it also catalyzed α-decarboxylation of aspartate to a slight extent. A sharp pH optimum at pH 7.0 has been observed. Km values of 0.7 mm and 0.05 µm were found for glutamate and pyridoxal phosphate, respectively. The enzyme was dissociated into two physically indistinguishable subunits with a molecular weight of 44,000 ± 2,000 in 6 m guanidine HCl containing 0.1 m β-mercaptoethanol.