Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla.
A high affinity binding site for [3H]dihydrotetrabenazine is thought to be present on the monoamine transport protein from chromaffin granules. We describe a procedure for purification of this binding activity from frozen bovine adrenal tissue, and we partially characterize the purified preparation. Binding activity solubilized with sodium cholate and soybean lecithin was fractionated on wheat germ lectin-Sepharose, phenyl-Sepharose, Mono Q, and hydroxylapatite. Denaturing electrophoresis of the purified binding activity, followed by silver staining, revealed a single broad band centered at an apparent molecular weight of 85,000. This preparation bound [3H]dihydrotetrabenazine with an apparent dissociation constant of 2.7 nM and had a site density of 10 nmol/mg. Treatment of the purified protein with neuraminidase reduced the apparent molecular weight by 9000, indicating the presence of terminal sialic acids on the oligosaccharide portion of this molecule.
Vincent
M S
MS
Pharmacology Section, Indiana University School of Medicine, Bloomington 47405.
Near
J A
JA
D000313:Adrenal Medulla / Q000737:chemistry*; D000818:Animals; D000837:Anion Exchange Resins; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D012117:Resins, Synthetic; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate; D013747:Tetrabenazine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D014316:Tritium; D014909:Wheat Germ Agglutinins
D000837:Anion Exchange Resins; D002352:Carrier Proteins; D006882:Hydroxyapatites; D012117:Resins, Synthetic; D014909:Wheat Germ Agglutinins; D014316:Tritium; C032811:dihydrotetrabenazine; D012967:Sodium Dodecyl Sulfate; C042855:Mono Q; D012685:Sepharose; D017886:Durapatite; D013747:Tetrabenazine