Solubilization and molecular characterization of active pancreastatin receptors from rat liver membranes.
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 Kd 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 Mr. Gel filtration of soluble extracts on Sephacryl S-300 revealed two molecular components with binding abilities (Mr 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 Mr, suggesting an association of the receptor with a G protein. Moreover, direct evidence that a Gq 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 Mr glycoprotein associated with G proteins.
Sánchez-Margalet
V
V
Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, University of Seville, Spain. Vsanchez@cica.es
Santos-Alvarez
J
J
D000818:Animals; D053379:Chromogranin A; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008566:Membranes / Q000648:ultrastructure; D008970:Molecular Weight; D010187:Pancreatic Hormones / Q000378:metabolism; D010191:Pancreatic Polypeptide / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
D053379:Chromogranin A; D006023:Glycoproteins; D010187:Pancreatic Hormones; D011964:Receptors, Gastrointestinal Hormone; C029438:receptors, pancreatic polypeptide-specific; C050836:pancreastatin; D010191:Pancreatic Polypeptide