Synaptic vesicles from hog brain-their isolation and the coupling between synthesis and uptake of ?-aminobutyrate by glutamate decarboxylase

Abstract

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of ?-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively. Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca(2+). Magnesium in concentrations 0.2-10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca(2+), but not Mg(2+), induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca(2+). Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca(2+). The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [(3)H]GABA, newly synthetized from [(3)H]glutamate, and the uptake of added [(14)C]GABA. No significant uptake of [(14)C]GABA was found under the experimental conditions used, whereas large amounts of [(3)H]GABA were found within the vesicles. It appears that the [(3)H]GABA accumulation process is functionally linked to [(3)H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.