Stereospecific (−)-[3H]norepinephrine binding to bovine hypothalamus possible identification of the catecholamine uptake site in synaptic vesicles

A (?)-³H]norepinephrine binding site was identified in a crude synaptosomal fraction isolated from bovine hypothalamus which bound norepinephrine rapidly, reversibly, and stereospecifically. The results were most consistent with binding of (-)-³H]norepinephrine to the carrier molecule used to translocate biogenic amines into synaptic vesicles. The binding studies indicated that specific binding of (?)-³H]norepinephrine to the crude synaptosomal fraction was greatly enhanced by 1 mM MgCl₂ and 1 mM ATP. The increased binding of (?)-³H]norepinephrine also occured in the presence of MgCl₂ and GTP, but AMP, adenosine and adenyl-5′-yl imidodiphosphate would not substitute for ATP. Neither CaCl₂ nor ZnSO₄ could be substituted for the MgCl₂. In the presence of MgCl₂ and ATP, the dissociation constant for (?)-³H]norepinephrine was 280 nM with a specific binding site density of 4.8 pmol/mg protein. Binding was stereospecific with ratios of 15, 4, and 6.5 for the affinities of (?)-isomers to (+)-isomers for norepinephrine, epinephrine and isoproterenol, respectively. Drug competition studies, conducted in the presence of Mg²⁺ and ATP, indicated that (?)-epinephrine, (?)-norepinephrine, dopamine and serotonin had inhibitory constants ranging from 0.25 to 0.8 μM with (?)-isoproterenol and tyramine having inhibitory constants around 2 μM. Reserpine was the most potent inhibitor having an inhibition constant of 8.6 ± 0.3 nM. The binding data were not consistent with the specific site being the α- or β-receptors for norepinephrine, the Uptake₁ Site for norepinephrine into synaptosomes or the metabolizing enzymes for norepinephrine.