title: Solubilization of high affinity peptide-YY receptors from porcine brain.
abstract: We have previously characterized peptide-YY (PYY) receptors in porcine hippocampal membranes. We demonstrate here that brain PYY receptors can be extracted in the active state using digitonin. Among several detergents tested for their suitability to extract active PYY receptors, digitonin gave the most favorable results, as judged by specific binding of [125I]PYY to the solubilized receptors. The binding of [125I]PYY 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 membrane PYY receptor: PYY greater than neuropeptide Y (NPY) much much greater than avian and porcine pancreatic polypeptide, and PYY greater than PYY-(22-36) much much greater than 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 digitonin extract; the high affinity component had affinities and binding capacities 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, solubilized with digitonin, and cross-linked to digitonin-solubilized receptors. The resulting complexes were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Using these procedures, we identified a PYY receptor species with a molecular size of 50,000, which was the same size as the labeled protein in native membrane homogenates. Solubilized NPY receptors were also the same size. The solubilized cross-linked PYY receptor was adsorbed by wheat germ agglutinin-agarose and Concanavalin-A, suggesting its glycoprotein nature. These data suggest that the specific binding properties of the PYY receptor are inherent in the solubilized glycoprotein molecules. The solubilization in digitonin of PYY receptors from membranes should allow a more complete molecular and functional characterization of PYY-mediated events and purification of the receptor.
authors.affiliation: Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Inui
authors.forename: M
authors.initials: M
authors.lastname: Okita
authors.forename: M
authors.initials: M
authors.lastname: Miura
authors.forename: Y
authors.initials: Y
authors.lastname: Hirosue
authors.forename: M
authors.initials: M
authors.lastname: Nakajima
authors.forename: K
authors.initials: K
authors.lastname: Shii
authors.forename: S
authors.initials: S
authors.lastname: Baba
authors.forename: A
authors.initials: A
authors.lastname: Balasubramaniam
authors.forename: M
authors.initials: M
authors.lastname: Kasuga
mesh_terms: D000327:Adsorption; D000818:Animals; D002850:Chromatography, Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry*; D066298:In Vitro Techniques; D037102:Lectins / Q000378:metabolism; D008970:Molecular Weight; D009478:Neuropeptide Y / Q000378:metabolism / Q000494:pharmacology; D019894:Peptide YY; D010455:Peptides / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification*; D012995:Solubility; D013552:Swine
chemical_list: D037102:Lectins; D009478:Neuropeptide Y; D010455:Peptides; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D019894:Peptide YY; D016244:Guanosine 5'-O-(3-Thiotriphosphate)