Sievert M K, Ruoho A E
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J Biol Chem. 1997 Oct 10;272(41):26049-55. doi: 10.1074/jbc.272.41.26049.
The full-length cDNA for the rat recombinant synaptic vesicle monoamine transporter (rVMAT2) containing a COOH-terminal polyhistidine epitope was engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed to high levels and purified to >95% homogeneity using immobilized Ni2+-affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was photolabeled using [125I]-7-azido-8-iodoketanserin ([125I]AZIK) and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine ([125I]TBZ-AIPP). Both [125I]AZIK and [125I]TBZ-AIPP photoaffinity labeling of purified rVMAT2 were protectable by 10 microM tetrabenazine (TBZ), 10 microM 7-aminoketanserin, and 1 mM concentrations of the transporter substrates dopamine, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal microsequenced. Radiosequencing of [125I]AZIK-labeled rVMAT2 indicated derivatization of Lys-20 in the NH2 terminus, just prior to putative transmembrane domain 1 (TMD1). [125I]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxtaposition of TMD1 and 10/11.
将含有COOH末端多组氨酸表位的大鼠重组突触小泡单胺转运体（rVMAT2）的全长cDNA构建到杆状病毒DNA中，以便在草地贪夜蛾（Sf9）细胞中表达。利用这种重组杆状病毒和培养的Sf9细胞，rVMAT2已高水平表达，并通过固定化Ni2+亲和层析，随后用凝集素（伴刀豆球蛋白A）层析纯化至>95%的纯度。使用[125I]-7-叠氮基-8-碘酮色林（[125I]AZIK）和[125I]2-N-[(3'-碘-4'-叠氮基苯基)丙酰基]丁苯那嗪（[125I]TBZ-AIPP）对纯化的转运体进行光标记。纯化的rVMAT2的[125I]AZIK和[125I]TBZ-AIPP光亲和标记均可被10μM丁苯那嗪（TBZ）、10μM 7-氨基酮色林以及1 mM浓度的转运体底物多巴胺、去甲肾上腺素和血清素所保护。使用酶法和化学方法生成放射性标记的肽段，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行纯化，并对NH2末端进行微测序。[125I]AZIK标记的rVMAT2的放射测序表明，在假定的跨膜结构域1（TMD1）之前的NH2末端的Lys-20发生了衍生化。[125I]TBZ-AIPP在TMD10和11中Gly-408和Cys-431之间的rVMAT2片段上进行了衍生化。这些数据表明TMD1与10/11并列。
