Journal
ANALYTICAL BIOCHEMISTRY
Volume 367, Issue 1, Pages 68-78Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.04.046
Keywords
vesicle monoamine transporter; VMAT2; reserpine; tetrabenazine; photoaffinity labels
Funding
- NINDS NIH HHS [R01 NS033650, NS33650, R01 NS033650-11] Funding Source: Medline
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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)glycyljmethyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3 H]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 mu M for TBZ-AIPP. Carrier-free radioiodinated [I-125]IAPEGlyMER and [I-125]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [I-125]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 [I-125]IAPEGlyMER was blocked by 100 nM reserpine, 10 mu M tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [I-125]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 mu M reserpine and 10 mu M tetrabenazine. Incubation of [I-125]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 [I-125]IAPEGlyMER and [I-125]TBZ-AIPP are effective photoaffinity labels for VMAT2. (C) 2007 Elsevier Inc. All rights reserved.
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