HYDRODYNAMIC PROPERTIES OF MUSCARINIC ACETYLCHOLINE-RECEPTORS SOLUBILIZED FROM RAT FOREBRAIN

Muscarinic receptors from rat forebrain were solubilized by Lubrol PX, lysophosphatidylcholine (LPC), digitonin and cholate/1 M NaCl. The overall level of solubilization was characterized using receptors prelabeled with an irreversible antagonist. The recovery of non-denatured soluble binding activity was estimated using reversible tritiated antagonists [propylbenzilylcholine mustard, quinuclidinylbenzilate and N-methylscopolamine]. All these detergents solubilized 60-85% of the total binding sites. In Lubrol PX most of the receptors were recovered in a denatured form. In the other detergents 30-90% of the solubilized receptors were stable and capable of binding reversible [3H]-antagonists with high affinity. The hydrodynamic properties of the soluble receptors were examined by gel filtration and sucrose gradient centrifugation in H2O and D2O. The soluble receptors in Lubrol PX, lysophosphatidylcholine and cholate were, in general, heterogeneous as regards their molecular size. Estimates of MW after correction for bound detergent varied from 82,000 to 134,000. Conditions were identified under which the receptor was largely monodisperse and the estimates of MW agreed with values (.apprx. 83,000) from sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The amount of bound detergent could not be calculated for the digitonin-muscarinic receptor complex which had an estimated overall median MW of .apprx. 290,000. A subpopulation of muscarinic receptors from the rat forebrain is evidently capable of existing in a monomeric soluble form and binding ligands. There is also evidence that complexes with other proteins can exist, but their specificity and functional relevance are not known.