Sodium-independent binding of gamma aminobutyric acid (GABA) to receptor-like sites in mammalian brain homogenates was much greater in membrane fractions which had been thoroughly washed with buffer, or detergent, and frozen and thawed several times, than in fresh unwashed membranes. As previously shown (Greenlee, Van Ness, & Olsen, Life Sciences 22, 1653 (1978), the washing procedure removed endogenous inhibitors of GABA binding which led to an apparent improvement in GABA binding affinity to a low affinity class of sites (KD ≅ 170 nM), and, additionally, the appearance of a high affinity (KD ≅ 10 nM) class of sites. This endogenous inhibitory material was found to inhibit both classes of GABA binding sites, but with greater potency towards the high affinity sites for GABA. Biochemical characterization of the inhibitor fraction revealed that the activity was heat-stable, insensitive to trypsin and disulfide reducing compounds, dialyzeable through membrane sieves which would retain molecules with a molecular weight of 5000, and eluted 100% from a molecular sieve column in the position of small molecules (salt volume), clearly separated from a 16,000 molecular weight marker. The inhibitor was over 80% inactivated by the enzyme GABAse, indicating that most, and perhaps all of the endogenous inhibitor of GABA binding was indeed GABA itself. The difficulty in removing endogenous GABA from brain membranes must be considered in studies on benzodiazepine receptors (since they are affected in vitro by GABA) and in any comparison of GABA or benzodiazepine receptors in human neuropsychiatric disorders, drug treatment or lesion studies.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Pharmacology, Toxicology and Pharmaceutics(all)