Molecular size of [3H]WB-4101-binding sites in rat cortex as determined by radiation inactivation

The molecular weight of the [3H]WB-4101-binding sites in rat cerebral cortex was estimated by the irradiation-inactivation technique. The molecular weight was found to be dependent on the assay concentrations of the radioligand in the binding assay. Assays with a [3H]WB-4101 concentration of 0.25 nM showed a molecular weight of 62,100 daltons and 5.1 nM showed 50,800 daltons. Scatchard transformation of the [3H]WB-4101-binding data shows two binding sites (high-affinity: KD = 0.09 nM, Bmax = 9.1 pmoles/g; low-affinity: KD = 20 nM, Bmax = 80 pmoles/g). It is suggested that the two binding exist at two distinct molecules and in that case the observed molecular weights of 62,100 and 50,800 daltons are not true values because the determinations are carried out on a mixture of the two molecule populations. The distribution of the two binding sites was calculated for the two radioligand concentrations, 0.25 nM and 5.1 nM; and on this background the "true" molecular weights of the two [3H]WB-4101-binding sites were estimated to be 68,300 daltons for the high-affinity molecule and 41,400 daltons for the low-affinity molecule. Competition studies with a variety of adrenergic agonists and antagonists against [3H]WB-4101 supported the hypothesis that only the high-affinity binding site is an alpha-1-adrenoceptor.