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J. Biol. Chem., Vol. 266, Issue 17, 10762-10767, 06, 1991

Solubilization and hydrodynamic properties of active peptide YY receptor from rat jejunal crypts. Characterization as a Mr 44,000 glycoprotein

T Voisin, A Couvineau, C Rouyer-Fessard and M Laburthe
Unite de Recherche sur la Differenciation et la Neuroendocrinologie de Cellules Digestives, Institut National de la Sante et de la Recherche Medicale, Villejuif, France.

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.
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