Solubilization of the catecholamine carrier of chromaffin granule membranes in a form that binds substrates and inhibitors of uptake.
The component of bovine chromaffin granule membrane responsible for the binding of [2-3H]dihydrotetrabenazine has been solubilized by treatment with detergents. The binding activity of the soluble material was measured either by equilibrium dialysis or by precipitation of the protein by poly(ethylene glycol) and filtration of the precipitate. The best yields and activities were obtained with sodium cholate, which solubilized more than 70% of the binding sites; deoxycholate had deleterious effects on the soluble activity, and among nonionic detergents, octyl beta-glucoside was the most efficient. With cholate, [3H]dihydrotetrabenazine binding occurred on soluble sites as judged by centrifugation of the bound material and by filtration on Sepharose 6B columns. Binding could be measured in the presence of cholate, but high detergent concentrations had a reversible inhibitory effect. The [3H]dihydrotetrabenazine binding characteristics of the soluble material were similar to those of the granule membrane, with one class of binding sites (KD = 23 nM, Bmax = 90 pmol/mg of protein). The association and dissociation rate constants were 0.072 X 10(6) M-1 s-1 and 1.1 X 10(-3) s-1, respectively. The pharmacological properties of the binding sites were also similar to those of the membranes. Our results thus support the view that the catecholamine carrier has been solubilized in an active conformation that binds substrates and inhibitors of uptake.
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050482:Catecholamine Plasma Membrane Transport Proteins; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins; D012995:Solubility; D013747:Tetrabenazine / Q000031:analogs & derivatives / Q000378:metabolism
D002352:Carrier Proteins; D050482:Catecholamine Plasma Membrane Transport Proteins; D002395:Catecholamines; D003902:Detergents; D026901:Membrane Transport Proteins; C032811:dihydrotetrabenazine; D013747:Tetrabenazine