Affinity purification of pancreastatin receptor-Gq/11 protein complex from rat liver membranes.
Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein.
Santos-Alvarez
J
J
Department of Medical Biochemistry and Molecular Biology, School of Medicine, Virgen Macarena University Hospital, Seville, Spain.
Sánchez-Margalet
V
V
D000818:Animals; D002846:Chromatography, Affinity; D053379:Chromogranin A; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000737:chemistry*; D008566:Membranes / Q000737:chemistry; D008970:Molecular Weight; D010187:Pancreatic Hormones / Q000378:metabolism; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D053379:Chromogranin A; D010187:Pancreatic Hormones; D011964:Receptors, Gastrointestinal Hormone; C029438:receptors, pancreatic polypeptide-specific; C050836:pancreastatin; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11