Specific binding of .BETA.-endorphin to the isolated renal basolateral membranes in vitro.

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Abstract
Binding of human β-endorphin (β-EP) to rat renal basolateral membranes was characterized using [125I]Try27-β-EP ([125I]β-EP) as a primary ligand. Ten millimolar of ethylenediaminetetra acetic acid (EDTA) completely inhibited the degradation of [125I]β-EP in the incubation mixture at 4°C, thus making it possible to quantitatively examine the [125I]β-EP binding. The specific binding of [125I]β-EP to the basolateral membranes was reversible and saturable, and a nonlinear least-squares regression analysis of a saturation isotherm revealed two different classes of specific binding sites. One class had an apparent dissociation constant (Kd) of 0.68nM and a lower number of binding sites (33fmol/mg protein), whereas the other class had a lower affinity (apparent Kd of 210nM) and a higher number of binding sites (7.3pmol/mg protein). Inhibition of the [125I]β-EP binding by naloxone (10μM) was approximately only 20%, and that by D-Ala2-D-Leu5-enkephalin (10μM) was null, suggesting the major role of a non-opioid binding component in specific [125I]β-EP binding to basolateral membranes. Moreover, a 50% inhibition by 10μM of dynorphin(1-13) suggests that a certain region of the primary structure of β-EP, excluding at least the NH2-terminal enkephalin sequence, is of particular importance for the [125I]β-EP binding. These lines of evidence suggest the existence of two different classes of specific binding sites for β-EP on the renal basolateral membranes, and the high- and low-affinity bindings may be attributed to opioid and non-opioid receptors, respectively, as judged by known characteristics of opioid and non-opioid receptors in other peripheral tissues.

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