title: Specific derivatization of the vesicle monoamine transporter with novel carrier-free radioiodinated reserpine and tetrabenazine photoaffinity labels.
abstract: Two iodophenylazide derivatives of reserpine and one iodophenylazide derivative of tetrabenazine have been synthesized and characterized as photoaffinity labels of the vesicle monoamine transporter (VMAT2). These compounds are 18-O-[3-(3'-iodo-4'-azidophenyl)-propionyl]methyl reserpate (AIPPMER), 18-O-[N-(3'-iodo-4'-azidophenethyl)glycyl]methyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3H]dopamine uptake into purified chromaffin granule ghosts showed IC50 values of approximately 37 nM for reserpine, 83 nM for AIPPMER, 200 nM for IAPEGlyMER, and 2.1 microM for TBZ-AIPP. Carrier-free radioiodinated [125I]IAPEGlyMER and [125I]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [125I]IAPEGlyMER labeling of a polypeptide that migrated as a broad band (approximately 55-90 kDa), with the majority of the label located between 70 and 80 kDa. The labeling by [125I]IAPEGlyMER was blocked by 100 nM reserpine, 10 microM tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [125I]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 microM reserpine and 10 microM tetrabenazine. Incubation of [125I]TBZ-AIPP-photolabeled chromaffin granule membranes in the presence of the glycosidase N-glycanase shifted the apparent molecular weight of VMAT2 to approximately 51 kDa. These data indicate that [125I]IAPEGlyMER and [125I]TBZ-AIPP are effective photoaffinity labels for VMAT2.
authors.affiliation: Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Sievert
authors.forename: Abdol R
authors.initials: AR
authors.lastname: Hajipour
authors.forename: Arnold E
authors.initials: AE
authors.lastname: Ruoho
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D012110:Reserpine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D012701:Serotonin / Q000378:metabolism; D013747:Tetrabenazine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D050493:Vesicular Monoamine Transport Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D007457:Iodine Radioisotopes; D050493:Vesicular Monoamine Transport Proteins; D012701:Serotonin; D012110:Reserpine; D013747:Tetrabenazine