Solubilization and Characterization of Muscarinic Acetylcholine Receptors

Abstract

Three distinct types of detergent have been found to be capable of solubilizing muscarinic binding activity from the rat forebrain. These are: (a) the alkylpolyoxyethylenes such as Lubrol PX [1]; (b) steroids such as cholate [2, 3], digitonin [4, 5] and the cholate sulphobetaine derivative CHAPS [6]; and (c) lysophosphatidylcholine [7]. In such studies, it has proved convenient to compare the overall yield of solubilized receptors from brain membranes (P₂ and P₃) pre-labelled with the irreversible alkylating affinity label [³H]propylbenzilylcholine (³H-PrBCM) [8] with the yield of active binding sites which can be assayed in the supernatant after extraction of non-pre-labelled membranes. Solubilized binding sites were conveniently measured by incubating 600,000 g-min supernatants with receptor-saturating concentrations (3 × 10^-8 M) of (-)-N-[Me-³H]- scopolamine (³H-NMS) or (-)-[³H]quinuclidinyl-benzilate (³H-QNB), followed by separation of bound from free ligand by rapid gel filtration on small columns of Sephadex G-50 M [3].