Solubilization and hydrodynamic properties of active peptide YY receptor from rat jejunal crypts. Characterization as a Mr 44,000 glycoprotein.
Peptide YY (PYY) receptors were solubilized from rat jejunal crypts using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS). The binding of [125I-Tyr36]monoiodo-PYY ([125I]PYY) to CHAPS extracts was time-dependent and reversible. The order of potency of PYY-related peptides for inhibiting [125I]PYY binding was PYY greater than neuropeptide Y much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data indicated the presence in soluble extracts of a single class of binding sites with a Kd of 1.02 +/- 0.26 nM and a Bmax of 79 +/- 6 fmol/mg protein. Gel filtration on Sephacryl S-300 and ultracentrifugation on sucrose density gradients of soluble [125I] PYY-receptor complexes revealed a single binding component with the following hydrodynamic parameters: Stokes radius, 4.43 nm; s20,w, 2.48 S; Mr, 48,000; frictional ratio, 1.82. Solubilized PYY receptors bound specifically to concanavalin A-, wheat germ agglutinin-, and soybean-coupled Sepharose, supporting their glycoproteic nature. After cross-linking with disuccinimidyl suberate, electrophoresis of covalent [125I]PYY-receptor complexes in membranes or CHAPS extracts revealed the presence of two bands of Mr 49,000 or 28,000 whose labeling was completely abolished by 1 microM unlabeled PYY. The Mr 49,000 band probably corresponded to the Mr 48,000 PYY-receptor complex evidenced by hydrodynamic studies. Assuming one molecule of [125I]PYY (Mr 4,000) was bound per molecule of receptor, these data show that intestinal PYY receptor consists of a Mr 44,000 glycoprotein after solubilization with CHAPS. The availability of this CHAPS-soluble receptor from rat jejunum represents a major step toward the purification of this newly characterized receptor.
Voisin
T
T
Unité de Recherche sur la Différenciation et la Neuroendocrinologie de Cellules Digestives, Institut National de la Santé et de la Recherche Médicale, Villejuif, France.
Couvineau
A
A
Rouyer-Fessard
C
C
Laburthe
M
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004848:Epithelium / Q000378:metabolism; D007583:Jejunum / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D019894:Peptide YY; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D002793:Cholic Acids; D010455:Peptides; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D019894:Peptide YY; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate