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

Abstract

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.). 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. 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. 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 (greater than 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). 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.