PEPTIDE-MAPPING OF THE [I-125] IODOAZIDOKETANSERIN AND [I-125] N-[(3'-IODO-4'-AZIDOPHENYL)PROPIONYL]TETRABENAZINE BINDING-SITES FOR THE SYNAPTIC VESICLE MONOAMINE TRANSPORTER
Mk. Sievert et Ae. Ruoho, PEPTIDE-MAPPING OF THE [I-125] IODOAZIDOKETANSERIN AND [I-125] N-[(3'-IODO-4'-AZIDOPHENYL)PROPIONYL]TETRABENAZINE BINDING-SITES FOR THE SYNAPTIC VESICLE MONOAMINE TRANSPORTER, The Journal of biological chemistry, 272(41), 1997, pp. 26049-26055
The full-length cDNA for the rat recombinant synaptic vesicle monoamin
e transporter (rVMAT2) containing a COOH-terminal polyhistidine epitop
e was engineered into baculovirus DNA for expression in Spodoptera fru
giperda (Sf9) cells. Using this recombinant baculovirus and cultured S
f9 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 ph
otolabeled using [I-125]-7-azido-8-iodoketanserin ([I-125]AZIK) and -[
(3'-iodo-4'-azido-phenyl)propionyl]tetrabenazine ([I-125]TBZ-AIPP). Bo
th [I-125]AZIK [I-125]TBZ-AIPP photoaffinity labeling of purified rVMA
T2 were protectable by 10 mu M tetrabenazine (TBZ), 10 mu M 7-aminoket
anserin, and 1 mM concentrations of the transporter substrates dopamin
e, norepinephrine, and serotonin. Radiolabeled peptides were generated
using enzymatic and chemical methods, purified using sodium dodecyl s
ulfate-polyacrylamide gel electrophoresis, and NH2-terminal microseque
nced. Radiosequencing of [I-125]AZIK-labeled rVMAT2 indicated derivati
zation of Lys-20 in the NH2 terminus, just prior to putative transmemb
rane domain 1 (TMD1). [I-125]TBZ-AIPP derivatized a segment of rVMAT2
between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxt
aposition of TMD1 and 10/11.