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Differential Solubilization of γ‐Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain
Author(s) -
Lester Bruce R.,
Miller Ann L.,
Peck Ernest J.
Publication year - 1981
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1981.tb02390.x
Subject(s) - chemistry , sodium , membrane , binding site , biophysics , biochemistry , biology , organic chemistry
Sodium‐dependent (+Na) and sodium‐independent (‐Na) receptive sites for γ‐aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X‐100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with Na receptive sites whether assayed in the presence or absence of 100 mwNaC1. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants, K D and B max , for –Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, while in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the K D was reduced to 60 ± 6.9 nM and the B max was reduced to 2.5 ± 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor, and not to inhibition by residual Triton X‐100. Multiple extraction with 0.1% or 0.5% Triton X‐100 did not alter the K B or B max , values for the binding of [ 3 H] GABA to JC. Sodium‐independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.