Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-( sup 125 I)iodoketanserin

Journal Article · Tue Mar 07 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00431a044· OSTI ID:5405146

Isambert, M F; Gasnier, B; Laduron, P M; Henry, J P ^[1]

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1. Institut de Biologie Physico-Chimique, Paris (France)

An iodinated azido derivative of ketanserin, 7-azido-8-(125I)iodoketanserin ( (125I)AZIK), has been used to label the monoamine transporter of bovine chromaffin granule membranes by the technique of photoaffinity labeling. In the dark, this derivative was found to bind reversibly to the membranes, with an equilibrium dissociation constant estimated to be 6 nM at 0 degrees C. As for ketanserin, binding occurred at the tetrabenazine site: (i) (125I)AZIK was displaced efficiently from its binding site by tetrabenazine, ketanserin, and 7-azidoketanserin, whereas serotonin, which is a substrate for the transporter but has a low affinity for tetrabenazine binding site, was a poor displacer; pipamperone and pyrilamine, two antagonists of respectively serotonin S2 and histamine H1 receptors, were inactive. (ii) 7-Azidoketanserin was a competitive inhibitor of (3H)dihydrotetrabenazine binding, and it inhibited the ATP-dependent uptake of serotonin by chromaffin granule ghosts. Irradiation of (125I)AZIK with long-wavelength UV light, followed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and autoradiography, revealed irreversible labeling of a membrane component with an apparent molecular weight of 73,000. Tetrabenazine inhibited the labeling of this 73-kDa band in a manner parallel to the binding of (125I)AZIK in the dark. Such a labeling is totally compatible with previous results obtained through photolabeling with a tetrabenazine derivative or by target size analysis. Moreover, preliminary experiments showed that (125I)AZIK can label the tetrabenazine binding sites of various sources including rat striatum, rabbit platelets, human pheochromocytoma, and human adrenal medulla. Therefore, this molecule appears to be an excellent probe to label the monoamine transporter of different amine storage vesicles even without purification.

• OSTI ID: 5405146
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:5; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ABSORPTION
AFFINITY
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AUTORADIOGRAPHY
AZAARENES
AZIDES
AZOLES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
DRUGS
ELECTROMAGNETIC RADIATION
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
INDOLES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LABELLING
MAMMALS
MAN
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MEMBRANES
MOLECULAR WEIGHT
NEUROREGULATORS
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PRIMATES
PROTEINS
PYRROLES
RABBITS
RADIATIONS
RADIOISOTOPES
RADIOPROTECTIVE SUBSTANCES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
RUMINANTS
SEROTONIN
SPECIFICITY
SYMPATHOMIMETICS
TRITIUM COMPOUNDS
TRYPTAMINES
ULTRAVIOLET RADIATION
VERTEBRATES
VISIBLE RADIATION