Solubilized Muscarine Receptor
Date of Award
Doctor of Philosophy (Ph.D.)
Muscarine receptors of rat forebrain and brainstem were labeled with tritiated quinuclidinyl benzilate (QNB) and solubilized in Lubrol-PX and digitonin. Lubrol extracts 96% of the QNB binding sites from both brain areas. Digitonin solubilizes 75% of the muscarine receptors from forebrain and 55% from brainstem. Digitonin and Lubrol extraction of muscarine receptors is dependent upon detergent/protein ratio; optimal extraction was obtained using 2 mg digitonin/mg protein and 4 mg Lubrol/mg protein.Association of QNB with muscarine receptors occurs with a T(, 1/2) of 1 minute in both forebrain and brainstem membranes extracted in digitonin. The T(, 1/2) of dissociation is 96 minutes in the forebrain and 42 minutes in the brainstem. The kinetically derived dissociation constants are 9 pM for forebrain muscarine receptors and 25 pM for brainstem receptors.Scopolamine inhibits QNB binding to digitonin-solubilized muscarine receptors with an IC(,50) of 0.3 nM in forebrain and 0.2 nM in brainstem. Apparent K(,D) values are 0.8 in forebrain and 3.6 nM in brainstem. These values are close to those reported for intact membranes. Scopolamine inhibition of QNB binding follows mass action behavior. Carbachol inhibition of QNB binding deviates from simple mass action behavior, but IC(,50) values (0.3 mM forebrain; 0.1 mM brainstem) and apparent K(,D)'s (25 M forebrain; 2 M brainstem) are similar to those reported for membrane preparations. The presence of Tris-Mn('++) during incubation increases the affinity of digitonin-extracted muscarine receptors of rat forebrain from 24 M to 0.45 M. Thus, the digitonin-solubilized receptor system displays binding parameters similar to those of intact membranes, with respect to scopolamine, carbachol and QNB.Gel filtration studies demonstrate that Lubrol- and digitonin-extracted (('3)H)-QNB prelabeled binding sites of rat forebrain and brainstem elute as a single symmetrical peak which co-migrates with marker proteins having Stokes radii of 7 nm ((beta)-galactosidase and nicotine receptor dimer).Differential and density gradient centrifugation studies show that QNB binding sites follow patterns of distribution expected of proteins located at the synapse. Isolation of synaptosomal preparations by these techniques yields an approximate 30-fold purification over whole membrane homogenates.
Health Sciences, Pharmacology
Kalinoski, Darline Lynn, "Solubilized Muscarine Receptor" (1982). Dissertations from ProQuest. 1301.