[125I]NPY bound to a single class of saturable binding sites on bovine hippocampus membranes with a KD of 0.1 nM and Bmax of 165 fmol/mg of protein. The rank order of potency of NPY fragments and other structurally related peptides to inhibit [125I]NPY binding was: PYY ≥ NPY ≫ BPP ≥ APP and NPY > NPY-(13-36) > NPY-(18- 36) ≥ NPY-(20-36) ≫ NPY-(26-36) > NPY-(free acid). The identity of the NPY binding site was investigated by affinity labeling. Gel electrophoresis followed by autoradiography revealed a band with a mol mass of 50 kDa. Unlabeled NPY or PYY, but not BPP, HPP and APP, inhibited labeling of [125I]NPY to the 50 kDa protein band. Moreover, labeling was inhibited by NPY > NPY- (18-36) ≥ NPY-(13-36) ≥ NPY-(20-36) > NPY-(26-36) > NPY- (free acid). The binding of [125I]NPY and the intensity of the cross-linked band were reduced in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.7 nM and 0.6 nM, respectively). These studies demonstrate that bovine hippocampal membranes contain a 50 kDa [125I]NPY binding site that has the ligand specificity characteristic of the Y2 receptor subtype.
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