SOLUBILIZATION OF HIGH-AFFINITY PEPTIDE-YY RECEPTORS FROM PORCINE BRAIN

We have previously characterized peptide-YY (PYY) receptors in porcine hippocampal membranes. We demonstrate here that brain PYY receptors c an be extracted in the active state using digitonin. Among several det ergents tested for their suitability to extract active PYY receptors, digitonin gave the most favorable results, as judged by specific bindi ng of [I-125]PYY to the solubilized receptors. The binding of [I-125]P YY to digitonin extract was dependent on incubation time, temperature, and protein and magnesium ion concentrations and had a pH optimum of 6-7. Solubilized PYY receptors maintained the rank order of potencies for various related peptides and PYY fragments characteristic of the m embrane PYY receptor: PYY > neuropeptide Y (NPY) >>> avian and porcine pancreatic polypeptide, and PYY > PYY-(22-36) >>> PYY-(1-22) and PYY- (22-28), respectively. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitoni n extract; the high affinity component had affinities and binding capa cities similar to those of the membrane PYY receptor. Solubilized PYY receptors also retained their sensitivity to guanine nucleotides. PYY was cross-linked to its receptors with disuccinimidyl suberate, solubi lized with digitonin, and cross-linked to digitonin-solubilized recept ors. The resulting complexes were analyzed on sodium dodecyl sulfate-p olyacrylamide gel electrophoresis, followed by autoradiography. Using these procedures, we identified a PYY receptor species with a molecula r size of 50,000, which was the same size as the labeled protein in na tive membrane homogenates. Solubilized NPY receptors were also the sam e size. The solubilized cross-linked PYY receptor was adsorbed by whea t germ agglutinin-agarose and Concanavalin-A, suggesting its glycoprot ein nature. These data suggest that the specific binding properties of the PYY receptor are inherent in the solubilized glycoprotein molecul es. The solubilization in digitonin of PYY receptors from membranes sh ould allow a more complete molecular and functional characterization o f PYY-mediated events and purification of the receptor.