Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine.
Characterization of the catecholamine transporter in chromaffin granule membranes has been hampered by the lack of a radioligand with high specific activity which binds selectively to the carrier with high affinity. We report here the identification of a high affinity binding site for [3H]reserpine on chromaffin granule membranes isolated from bovine adrenal gland which has the characteristics expected of the catecholamine transporter. [3H]Reserpine bound predominately to a high affinity site with a Kd for [3H]reserpine of 9 nM and a binding site density of 7.8 pmol/mg of protein. Comparison of the characteristics of the high affinity reserpine binding site to the characteristics of catecholamine transport indicated that (a) the Ki and rank order of potency for inhibition of [3H]reserpine binding by various biogenic amines was similar to their Ki for inhibition of catecholamine transport (b) both the inhibition of (-)-[3H]norepinephrine transport and inhibition of [3H]reserpine binding showed similar stereo-specificity, and (c) Kd for binding of reserpine to chromaffin granule membranes was similar to the Ki for reserpine inhibition of catecholamine transport. These results demonstrate that the high affinity binding site for [3H]reserpine on chromaffin granule membranes is associated with the catecholamine transporter.
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050482:Catecholamine Plasma Membrane Transport Proteins; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins; D012110:Reserpine / Q000378:metabolism*
D002352:Carrier Proteins; D050482:Catecholamine Plasma Membrane Transport Proteins; D002395:Catecholamines; D026901:Membrane Transport Proteins; D012110:Reserpine