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Molecular characterization of muscarinic receptor subtypes in bovine cerebral cortex by radiation inactivation and molecular exclusion h.p.l.c.

1 Muscarinic receptor subtypes in bovine cerebral cortex were investigated by means of radiation inactivation and molecular exclusion high performance liquid chromatography (h.p.l.c). 2 The functional molecular size of the muscarinic receptor in situ was determined by the radiation inactivation method. The value for the muscarinic receptor labelled with [3H]‐quinuclidinylbenzilate ([3H]‐QNB) was 91,000 daltons, while that labelled with [3H]‐pirenzepine ([3H]‐PZ) was 157,000 daltons. 3 The muscarinic receptor solubilized with digitonin could be labelled with [3H]‐PZ as well as with [3H]‐QNB. 3‐[(3‐Cholamidopropyl)‐dimethylammonio] ‐ propane sulphonate (CHAPS) solubilized the muscarinic receptor labelled with [3H]‐QNB but not that labelled with [3H]‐PZ, in agreement with the low affinity of pirenzepine for inhibiting [3H]‐QNB binding in CHAPS‐solubilized preparations. 4 The size of the muscarinic receptor in solution was estimated by molecular exclusion h.p.l.c. The digitonin‐solubilized muscarinic receptor had a molecular weight of 290,000 and the [3H]‐QNB and [3H]‐PZ binding activities behaved identically. The CHAPS‐solubilized muscarinic receptor labelled with [3H]‐QNB was apparently of high molecular weight (> 1,000,000 Mr), indicating the formation of aggregates and/or micelles. In the presence of digitonin this form was dissociated into a lower molecular weight species (580,000 Mr). 5 These data indicate that the ligand binding component of the muscarinic receptor species labelled by both [3H]‐QNB and [3H]‐PZ exists on the same receptor protein, but that the [3H]‐PZ binding component in situ is probably coupled to other components in the membrane.