|dc.description.abstract||L-glutamate decarboxylase (GAD, EC.22.214.171.124), 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.126.96.36.199), 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.||en