Distance between the propylbenzilylcholine mustard attachment site and carbohydrates and thiol groups in muscarinic acetylcholine receptor protein from rat cerebral cortex

Abstract

When rat cerebral-cortex membranes were labelled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), a single protein of Mr 68,000 was found to carry the atropine-sensitive covalent label. After trypsinolysis of the receptors solubilized in 0.075% SDS, the resulting fragments were submitted to size analysis in combination with wheat-germ agglutinin (WGA)-Sepharose and organomercurial-agarose chromatography. Peptides of Mr 75,000, 50,000, 30,000, 18,000 and 8000 were specifically released from the receptor. All fragments above Mr 8000 were able to bind WGA-Sepharose and therefore the peptide of Mr 18,000 was taken as the upper limit of the distance between the antagonist and the glycan moieties. The limit fragment of Mr 8000 carried chemical groups which were modified by N-ethylmaleimide and reacted with an immobilized organomercurial. About 65-80% of the labelled receptors were adsorbed on concanavalin A-Sepharose with low affinity, generating two further components after sequential application to WGA-Sepharose. About 50% of the receptors were susceptible to neuraminidase treatment, with a concomitant slight modification of the SDS/polyacrylamide-gel-electrophoretic pattern.