title: Detergent solubilization and affinity purification of a local anesthetic binding protein from mammalian axonal membranes.
abstract: Previously, we identified a local anesthetic receptor in the membranes of nerve cells, especially enriched in axonal membranes, by the binding assay of a fluorescence local anesthetic probe, quinacrine (Greenberg, M., and Tsong, T.Y. (1982) J. Biol. Chem. 257, 8964-8971). We report here affinity chromatography purification of the quinacrine binding protein from axonal membranes of bovine brain. One per cent sodium cholate solubilized more than 90% of the membrane proteins, and the quinacrine binding activity was preserved in the extract; KD remained at 1.0 microM. An affinity column of quinacrine mustard covalently linked to Affi-Gel 10 (Bio-Rad) retained about 5% of the total solubilized proteins, and the subsequent elution with 1.0 M lidocaine revealed a protein peak which coeluted with the lidocaine peak. After removal of lidocaine, the protein peak retained the high affinity to quinacrine (KD = 1.5 microM). Furthermore, bound quinacrine could be displaced with dibucaine in the range of its nerve-blocking concentration, around 10 microM. Sodium dodecyl sulfate-gel electrophoresis of the peak protein fractions gave a predominant band of approximately 16 kDa. A major protein band with the same molecular weight was also seen in the sodium dodecyl sulfate gel of the total axonal membrane proteins. The receptor concentration in the axonal membranes was estimated to be 2-5 nmol/mg protein. With a molecular weight of 16 kDa, the receptor should constitute 3-8% of total proteins, consistent with the finding that it is a major protein constituent of the axonal membranes. The purity of the binding protein after the affinity chromatography step was about 90%. The purified protein contained roughly 60 nmol of quinacrine binding sites/mg.
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D011796:Quinacrine / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D003902:Detergents; D011796:Quinacrine