Photoaffinity labeling of the tetrabenazine binding sites of bovine chromaffin granule membranes

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Abstract
An azido derivative of tetrabenazine, a specific inhibitor of the monoamine carrier of chromaffin granule membranes, was synthesized. In the dark, this compound, 3H-labeled N-(3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hdexahydro-11bH-benzo[.alpha.]quinolizin-2-yl)-4-[(4-azido-2-nitrophenyl)amino]butanamide [(3H]TBA), bound reversibly to purified chromaffin granule membranes. Centrifugation through SP-Sephadex columns was used to separate bound and free [3H]TBA. This technique gave low levels of nonspecific binding and allowed recovery of [3H]TBA-membrane complexes. Scatchard analysis of the data indicated 1 class of site with an equilibrium dissociation constant Kd of 50 nM and a density of sites of 40-50 pmol/mg of protein, consistent with reported densities of reserpine and dihydrotetrabenazine binding site. Competition experiments showed that TBA and tetrabenazine bound to the same site. Irradiation at 435 nm of [3H]TBA-membrane mixture induced some irreversible binding of the probe to membranes. After irreversible binding of TBA, the number of dihydrotetrabenazine binding sites was decreased, indicating that the probe was covalently bound to the monoamine carrier. [3H]TBA-membrane complexes isolated by centrifugation through SP-Sephadex columns were irradiated, and their radioactivity was analyzed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. A polypeptide with a MW of 70,000 was labeled. This polypeptide was different from dopamine-.beta.-hydroxylase, and it was not adsorbed on concanavalin A-Sepharose. The monoamine carrier of chromaffin granule membrane apparently has an oligomeric structure, involving a 45K subunit [R. Gabizon, T. Yetinson, and S. Schuldiner, (1982)] and a 70 K subunit.