Aspects of L-Glutamate decarboxylase and gaba metabolism in avian brain

Abstract

L-glutamate decarboxylase (GAD, EC.4.1.1.15), the enzyme responsible for the synthesis of the inhibitory neurotransmitter 4-aminobutyric acid (GABA) was partially purified from chick brain using a combination of column chromatographic (ion exchange, hydroxyapatite and gel filtration) and electrophoretic techniques. Preparative polyacrylamide gel electrophoresis resulted in enzyme inactivation and was, therefore, of limited use. Chick brain GAD proved to be unstable so attempts were made to develop an efficient affinity chromatography procedure (using the cofactor pyridoxal-5-phosphate (PLP) as the ligand) which would enable purification to homogeneity with a minimum number of steps. However, this technique depends on ready dissociation of PLP from the enzyme. Evidence is presented to suggest that the association between GAD and PLP is very tight and that dissociation does not occur easily. Chick brain GAD has an of approximately 150 000 +/- 15 000, calculated by gel filtration chromatography, and an apparent Km for its substrate glutamate of 3.7 +/- 0.4 mM. The localisation and regional variation of GABA metabolism in chick brain, from day 1 to day 14 post hatch, was investigated. activities of GAD and 4-aminobutyrate:2-oxoglutarate transaminase (GABA-T, EC.2.6.1.19), the enzyme responsible for the degradation of GABA, were determined in homogenates of tissue taken from six different brain regions. This was complemented by histochemical localisation of GABA-T in thin (20 urn) transversa sections of chick brain. It had been intended to localise GABAergic neurons by immunocytochemical techniques however, lack of a homogenous protein precluded conventional methods of raising antibodies in rabbits. Balb/c mice were injected with partially purified GAD and the spleen cells from an immune mouse fused with myeloma cells from the same strain of mouse. The hybrid cells produced were screened for anti-GAD activity. Two fusions were carried out in an attempt to raise monoclonal antibodies to chick brain GAD. From fusion 1, 3% of the wells seeded contained hybrids, and from fusion 2, 20% of the wells seeded contained hybrids. Preliminary screening experiments indicated that one of the hybrids formed from fusion 1 and several of those formed from fusion 2 may have secreted anti-GAD antibodies.