Characterization and purification of the monoamine transporter of bovine chromaffin granules.
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%.
Isambert
M F
MF
CNRS Unité Associée 1112, Institut de Biologie Physico-Chimique, Paris, France.
Gasnier
B
B
Botton
D
D
Henry
J P
JP
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000737:chemistry; D015306:Biogenic Monoamines / Q000378:metabolism*; D001692:Biological Transport; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007525:Isoelectric Focusing; D007650:Ketanserin / Q000031:analogs & derivatives / Q000737:chemistry; D009439:Neuraminidase / Q000378:metabolism; D010777:Photochemistry
D000345:Affinity Labels; D001386:Azides; D015306:Biogenic Monoamines; C067115:azidoiodoketanserin; D007650:Ketanserin; D009439:Neuraminidase