125I-Labeled alpha-bungarotoxin and [3H]quinuclidinylbenzilate binding to rat brain membranes. Effects of physical, chemical and enzymatic treatments.

The effects of various physical, chemical and enzymatic treatments of rat brain membranes were investigated with respect to 125I-labeled alpha-bungarotoxin ([125I]alpha BuTx) and [3H]quinuclidinylbenzilate ([3H]QNB) binding. Binding appeared relatively stable to autolysis, mechanical shearing, freeze-thawing, and divalent cation addition (Sr2+) or removal (EGTA, EDTA). Binding for [125I]-alpha BuTx was slightly reduced by trypsin digestion of the membranes while both [125I]alpha BuTx and [3H]QNB binding were reduced by phospholipase A2 digestion (Crotalus adamantus phospholipase A2 and beta-bungarotoxin). Treatment of the membranes with the disulfide reducing agent, dithiothreitol, resulted in additional [125I]alpha BuTx binding but showed little effect on [3H]QNB binding. Binding of the cholinergic agonists, nicotine and carbamylcholine, was studied by observing their concentration-dependent ability to inhibit [125I]alpha BuTx and [3H]QNB binding, respectively. Membrane sulfhydryl group reduction and endogenous cation removal by EGTA or EDTA resulted in a lowered affinity for nicotine bindng. Alkylation of membranes with N-ethylmaleimide resulted in an increase in carbamylcholine affinity. Other treatments had little or no effect on nicotine or carbamylcholine binding.