Abstract
In a medium without Na+, gamma-aminobutyric acid (GABA) binds at 0 degrees 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 degrees with two dissociation constants, 20 nM and 111 nM. The endogenous inhibitor is a thermostable (95 degrees for 15 min) acidic protein of approximately 1.5 X 10(4) 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 functional of GABA-ergic synapses is discussed.
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