Purification and reconstitution of the high affinity choline transporter.
The high-affinity choline transporter has been solubilized from synaptosomal membranes by various detergents. The solubilized carrier protein has been incorporated into liposomes after removal of the detergent by dialysis. Using the reconstitution of choline transport activity as an assay, the components catalyzing choline translocation were purified from the detergent extract by ion-exchange chromatography on a Mono-Q column followed by immunoaffinity chromatography. Monitoring the active fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectrofocussing gave one major protein with an apparent molecular weight of about 90,000 and an isoelectric point of pH 4.7. The isolated protein appeared to be heavily glycosylated as shown by lectin binding; upon treatment with endoglycosidase F the polypeptide was degraded to an apparent molecular weight of about 65,000. Accumulation of choline into liposomes reconstituted with the purified protein was driven by artificially imposed sodium gradients and inhibited by hemicholinium-3.
Knipper
M
M
University Stuttgart-Hohenheim, Institute of Zoophysiology, F.R.G.
Kahle
C
C
Breer
H
H
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002794:Choline / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006110:Grasshoppers; D006426:Hemicholinium 3 / Q000494:pharmacology; D007158:Immunologic Techniques; D007526:Isoelectric Point; D008081:Liposomes / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D012995:Solubility; D013570:Synaptic Membranes / Q000737:chemistry*
D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; D006426:Hemicholinium 3; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002794:Choline