Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate

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Abstract
The muscarinic cholinergic receptor present in synaptosomal membranes [SDS] of rat brain was covalently labeled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labeled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulfate [SDS], and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of SDS was analyzed. Provided both proteolysis and inter-chain disulfide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in SDS/polyacrylamide gel electrophoresis, as a broad band of apparent MW 66,000-76,000. Two dominantly labeled polypeptides, of apparent MW 68,000 and 73,000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labeled receptor readily formed intermolecular disulfide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulfide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent MW 119,000-128,000) occurred. This probably explains previous reports in the literature of larger MW species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulfide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5 % SDS with an equivalent Stokes'' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent MW in gel electrophoresis of 43,000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes'' radius, eluting just behind reduced bovine serum albumin (Stokes'' radius 8.5 nm; apparent MW in electrophoresis 67,000). Thus, the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behavior in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment. This does not support suggestions that the receptor is made from 2 smaller polypeptide chains held together by a hydroxylamine-labile linkage, such as an ester bond.