Purification of the vesamicol receptor.
The vesamicol receptor (VR) present in cholinergic synaptic vesicles isolated from the electric organ of Torpedo was solubilized in cholate detergent and stabilized with glycerol and a phospholipid mixture. The receptor was purified in 7% yield by hydroxylapatite, wheat germ lectin affinity, DEAE anion-exchange, and size exclusion chromatographies based on a [3H]vesamicol binding assay. A final specific binding of 4400 pmol/mg of protein was obtained. The cholate-solubilized VR complex exhibited variable aggregation states with particle molecular masses of 210-3500 kDa in different experiments. The purified VR exhibited very heterogeneous electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with very diffuse protein staining at about 240 kDa. No "classical" polypeptide or glycopeptide band was detected. One form of the SV1 epitope, which is characteristic of cholinergic synaptic vesicle proteoglycan, copurified precisely with the VR. The SV2 epitope, which is found in most neuronal and endocrine secretory vesicles, also closely purified with the VR. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site. This confirms the allosteric model for the VR in the AcCh transporter. The physicochemical properties of the VR and copurification of it with the SV1 epitope strongly suggest that the VR is associated with cholinergic vesicle proteoglycan. A second proteoglycan that is not associated with the VR but which carries the SV1 and SV2 epitopes also was observed.
Bahr
B A
BA
Department of Chemistry, University of California, Santa Barbara 93106.
Parsons
S M
SM
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D000495:Allosteric Site; D000818:Animals; D002845:Chromatography; D003902:Detergents; D004557:Electric Organ / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D010880:Piperidines / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017478:Receptors, Phencyclidine; D012995:Solubility; D013572:Synaptic Vesicles / Q000737:chemistry*; D014101:Torpedo
D003902:Detergents; D000939:Epitopes; D010880:Piperidines; D017981:Receptors, Neurotransmitter; D017478:Receptors, Phencyclidine; C006189:vesamicol; D000109:Acetylcholine