Solubilization of receptors for pancreatic polypeptide from rat liver membranes.
We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.
Nguyen
T D
TD
Division of Gastroenterology, Duke University, Durham, North Carolina.
Wolfe
M S
MS
Heintz
G G
GG
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D010191:Pancreatic Polypeptide / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility
D003432:Cross-Linking Reagents; D011964:Receptors, Gastrointestinal Hormone; C029438:receptors, pancreatic polypeptide-specific; D010191:Pancreatic Polypeptide