Characterization and purification of the monoamine transporter of bovine chromaffin granules

Abstract

The monoamine transporter of the chromaffin granule membranes can be specifically labeled by the photoaffinity reagent 7-azido-8-[125I]iodoketanserin. The characteristics of the labeled protein have been investigated. Two-dimensional gel electrophoresis of the labeled membranes indicated a MW of about 70,000 and an isoelectric point ranging from 3.8 to 4.6. No clear protein spot was associated with the radioactive material, which migrated between glycoproteins GPII and GPIV. The diffuse aspect of the radioactive material indicated a heterogeneity, which was not modified after a second electrophoresis. This heterogeneity was, at least partially, due to glycosylation of the transporter; neuraminidase treatment increased the protein pI up to 6.3, whereas digestion with N-glycopeptidase markedly decreased the apparent MW, from 70,000 to 50,000. SDS-polyacrylamide gel electrophoresis showed that, at low acrylamide concentrations, the labeled material migrated more rapidly than predicted from the mobility of the markers of molecular weight, a behavior which indicated a marked hydrophobicity of the transporter. The labeled protein was purified to homogeneity by a combination of chromatography on DEAE-cellulose at pH 4.5, on immobilized wheat germ agglutinin, and on hydroxylapatite in the presence of SDS. During this purification, the specific radioactivity was increased by a factor of 300-500, with a yield of 10-20%.

Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.

“…1 Also, in recent studies, Henry and collaborators (17) provided support for the above conclusion by analyzing the results of photoaffinity labeling with a derivative of tetrabenazine, another inhibitor of VAT: labeling of an 80-kDa polypeptide with a pI of around 3.5 was observed also by these investigators.…”

Expression and regulation of the bovine vesicular monoamine transporter gene

“…The vesicular monoamine transporter was purified from bovine chromaffin granules by the procedure of Isambert et al [5] and by a technique to be described (Gasnier, B., Sag& C. and Henry, J.P., in preparation). Peptides generated by CNBr cleavage were separated by reverse phase HPLC on an Aquapore RP-8 column, 100 x 2.1 mm (Brownlee).…”

“…Ketanserin, an antagonist of 5-HTz receptors, also binds to the same site as tetra~nazine [2]. These ligands have been used to purify the transporter in an active f3,4] or inactive form [5]. In addition, a pharmacological analysis of bovine chromaffin cells in culture suggested that the expression of the vesicular monoamine transporter is up-regulated under depolarizing conditions [6].…”

Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

“…Furthermore, photoaffinity labeling of storage vesicles from brain, adrenal medulla, and platelets from different species with 7-azido-8-[1251] (7)(8)(9).…”