| The nicotinic acetylcholine receptor of the moth, Manduca sexta, was characterized by monitoring {dollar}sp{lcub}125{rcub}{dollar}I-{dollar}alpha{dollar}-bungarotoxin binding to the crude homogenate supernate of the cerebral ganglion. Equilibrium binding, kinetic analysis of complex formation and the pharmacological specificity of competing ligands were investigated. Determination of the relative molecular mass (M{dollar}rmsb{lcub}r{rcub}){dollar} of the monomer and its component subunits was attempted. Polyacrylamide gel electrophoresis, under non-denaturing and denaturing conditions, and sucrose density gradient centrifugation were performed.; The toxin-receptor complex forms rapidly and exhibits an {dollar}rm Ksb{lcub}D app.{rcub}{dollar} of 1.3 nM. Kinetic studies demonstrate an association characterized by a {dollar}rm ksb1{dollar} or {dollar}6.5times10sp6{dollar} M{dollar}sp{lcub}-1{rcub}{dollar}' min{dollar}sp{lcub}-1{rcub}{dollar} and a dissociation characterized by a k{dollar}sb{lcub}-1{rcub}{dollar} or 0.014 min{dollar}sp{lcub}-1{rcub}.{dollar} The {dollar}rm Ksb{lcub}D{rcub}{dollar} calculated from these results, 2 nM, is in good agreement with the results of equilibrium binding studies. The dissociation is characterized by a rapidly dissociating moiety and a very slowly dissociating moiety. The {dollar}rm tsb{lcub}1/2{rcub}{dollar} for the former is 50 minutes.; The pharmacology of this receptor was nicotinic in nature as {dollar}alpha{dollar}-bungarotoxin, dihydro {dollar}beta{dollar} erythroidine, nicotine and curare were more effective competitors than muscarinic ligands. The binding site also displayed sensitivity to strychnine and bicuculline, ligands effective at the glycine and GABA receptors respectively. These receptors are classified in the same ligand-gated channel superfamily as the nicotinic acetylcholine receptor.; The nicotine competition binding profile was characterized by an unusually shallow area at mid-point in its binding curve. The shallowness is pronounced between 10{dollar}sp{lcub}-6{rcub}{dollar} M and 10{dollar}sp{lcub}-4{rcub}{dollar} M nicotine. This is similar to the range at which C. Morris (1984) determined Manduca nerve-cords were resistant as compared to Periplaneta nerve-cords to bath applied nicotine. The binding profiles of methyllycaconitine and dihydro {dollar}beta{dollar} erythroidine also displayed areas of unusual shallowness. This unusual interaction between the receptor and these ligands may reflect the molecular mechanism by which this organism tolerates high levels of nicotine in its calculating hemolymph.; The sedimentation coefficient of the monomer as determined by its migration in sucrose density gradient centrifugation is 10 S. This corresponds to a M{dollar}rmsb{lcub}r{rcub}{dollar} of about 300,000. The formation of larger aggregate was also observed. Electrophoresis under non-denaturing conditions demonstrated the aggregated protein and only in the presence of dithiothreitol was a protein of MT 325,000 seen. Affinity purified receptor migrated in non-denaturing gels as subunit-sized proteins and in denaturing gels as at Least two bands with {dollar}rm Msb{lcub}r{rcub}s{dollar} of 60,000 and 68,000. |
