Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Abstract

Muscarinic receptors from rat forebrain have been solubilized by Lubrol PX, lysophosphatidylcholine (LPC), digitonin and cholate/1 M sodium chloride. The overall level of solubilization was characterized using receptors prelabelled with an irreversible antagonist. The recovery of nondenatured soluble binding activity was estimated using reversible tritiated antagonists. 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 have been 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 the molecular weight 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 molecular weight agreed with values (ca. 83,000) from sodium dodecylsulphate (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 molecular weight of about 290,000. It is concluded that a subpopulation of muscarinic receptors from the rat forebrain is 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.