The effect of phospholipases and proteases on the binding of gamma-aminobutyric acid to junctional complexes of rat cerebellum.
A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with gamma-amino [14C]butyric acid at 25degreesC for 10 min, using [3H]sucrose as a marker for entrapped space, Total binding was determined in the absence of, and non-specific binding in the presence of, and excess of unlabelled gamma-aminobutyric acid. The difference bewteen the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or L-2,4-diaminobutyric acid. The above compounds had little effect on the non-specipic binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A2 (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with gamma-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with gamma-aminobutyric acid for binding to the receptor protein.
D000110:Acetylcholinesterase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000613:Aminobutyrates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002531:Cerebellum / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005649:Fumarate Hydratase / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D009708:Nucleotidases / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010740:Phospholipases / Q000494:pharmacology*; D011092:Polyethylene Glycols; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D012995:Solubility; D013569:Synapses / Q000187:drug effects / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000613:Aminobutyrates; D011092:Polyethylene Glycols; D005680:gamma-Aminobutyric Acid; D004492:Edetic Acid; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D010740:Phospholipases; D000110:Acetylcholinesterase; D009708:Nucleotidases; D010447:Peptide Hydrolases; D000251:Adenosine Triphosphatases; D005649:Fumarate Hydratase; D002118:Calcium