SPECIFIC BINDING OF BETA-ENDORPHIN TO THE ISOLATED RENAL BASOLATERAL MEMBRANES INVITRO

Binding of human beta-endorphin (beta-EP) to rat renal basolateral membranes was characterized using [I-125]Tyr27-beta-EP ([I-125]beta-EP) as a primary ligand. Ten millimolar of ethylened? iaminetetra acetic acid (EDTA) completely inhibited the degradation of [I-125]beta-EP in the incubation mixture at 4-degrees-C, thus making it possible to quantitatively examine the [I-125]beta-EP binding. The specific binding of [I-125]beta-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 0.68 nM and a lower number of binding sites (33 fmol/mg protein), whereas the other class had a lower affinity (apparent Kd of 210 nM) and a higher number of binding sites (7.3 pmol/mg protein). Inhibition of the [I-125]beta-EP binding by naloxone (10 mu-M) was approximately only 20%, and that by D-Ala2-D-Leu5-enkephalin (10 mu-M) was null, suggesting the major role of a non-opioid binding component in specific [I-125]beta-EP binding to basolateral membranes. Moreover, a 50% inhibition by 10 mu-M of dynorphin(1-13) suggests that a certain region of the primary structure of beta-EP, excluding at least the NH2-terminal enkephalin sequence, is of particular importance for the [125I]be[I-125]beta-EP binding. These lines of evidence suggest the existence of two different classes of specific binding sites for beta-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.