Purification of an endogenous protein inhibitor of the high affinity binding of gamma-aminobutyric acid to synaptic membranes of rat brain.

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Abstract
In a medium without Na+, GABA binds at 0.degree. C to freshly prepared crude synaptic membranes from rat cerebral cortex with an apparent dissociation constant of 218 nM. An endogenous inhibitor of the Na+-independent GABA binding was removed from these membranes by freezing and thawing and by repeated washing with Tris citrate buffer (50 mM, pH 7.1) containing 0.01% Triton X-100. As a result, the crude synaptic membranes bind GABA at 0.degree. C with 2 dissociation constants, 20 nM and 111 nM. The endogenous inhibitor is a thermostable (95.degree. C for 15 min) acidic protein of approximately 1.5 .times. 104 daltons. It was purified (about 500-fold) with a series of procedures including gel chromatography on Sephadex G-100 and ion exchange chromatography on Dowex 50W-X8 (H+). Recombination of the purified endogenous inhibitor with crude synaptic membrane preparations deprived of the endogenous inhibitor showed that the purified inhibitor blocked noncompetitively the sites for high-affinity GABA binding. A role of this endogenous regulator in the function of GABA-ergic synapses is discussed.