This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sánchez-Margalet, V. Articles by Santos-Álvarez, J. Search for Related Content PubMed PubMed Citation Articles by Sánchez-Margalet, V. Articles by Santos-Álvarez, J.

Solubilization and Molecular Characterization of Active Pancreastatin Receptors from Rat Liver Membranes¹

Department of Medical Biochemistry and Molecular Biology, Investigation Unit of Virgen Macarena Hospital, Faculty of Medicine, University of Seville, Seville, Spain

Address all correspondence and requests for reprints to: Dr. Víctor Sánchez-Margalet, Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla. Av. Sánchez Pizjuan 4, 41009 Seville, Spain. E-mail: Vsanchez{at}cica.es

Pancreastatin receptors were solubilized from rat liver membranes with the nonionic detergent Triton X-100. Binding of a iodinated analog of rat pancreastatin ([¹²⁵I-Tyr⁰]pancreastatin) to the soluble fraction was time dependent, saturable, and reversible. Scatchard analysis of binding under equilibrium conditions indicated that the soluble extracts contained a single class of pancreastatin-binding sites, with a binding capacity of 14 fmol/mg protein and a K_d of 0.3 nM. As observed with membrane-bound receptors, binding of [¹²⁵I]pancreastatin to soluble extracts was inhibited by guanine nucleotides with the following rank order of potency: guanyl-5'-yl-imidodiphosphate > GTP > GDP > GMP, indicating that the soluble receptors are functionally linked to G proteins. Molecular analysis of the soluble pancreastatin receptor by covalent cross-linking to [¹²⁵I]pancreastatin using disuccinimidyl suberate and further identification on SDS-PAGE indicated a single band of 85,000 M_r. Gel filtration of soluble extracts on Sephacryl S-300 revealed two molecular components with binding abilities (M_r 80,000 and 170,000). The higher molecular mass component was more sensitive to guanine nucleotides, and covalent cross-linking of both components to [¹²⁵I]pancreastatin and further SDS-PAGE analysis revealed again a single band of 85,000 M_r, suggesting an association of the receptor with a G protein. Moreover, direct evidence that a G_q was present in the same chromatographic fraction was obtained by specific immunodetection. The soluble receptor is a glycoprotein that can be specifically bound to the wheat-germ agglutinin lectin. We conclude that we solubilized active pancreastatin receptors from rat liver membranes, and these results support the conclusion that the liver pancreastatin receptor consists of a 80,000 M_r glycoprotein associated with G proteins.

This article has been cited by other articles:

				V. Sanchez-Margalet and C. Gonzalez-Yanes Pancreastatin inhibits insulin action in rat adipocytes Am J Physiol Endocrinol Metab, December 1, 1998; 275(6): E1055 - E1060. [Abstract] [Full Text] [PDF]

				D. T. Ward, E. M. Brown, and H. W. Harris Disulfide Bonds in the Extracellular Calcium-Polyvalent Cation-sensing Receptor Correlate with Dimer Formation and Its Response to Divalent Cations in Vitro J. Biol. Chem., June 5, 1998; 273(23): 14476 - 14483. [Abstract] [Full Text] [PDF]