title: Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor.
abstract: Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.
authors: Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
authors: R K
authors: RK
authors: Pearson
authors: L J
authors: LJ
authors: Miller
authors: E M
authors: EM
authors: Hadac
authors: S P
authors: SP
authors: Powers
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D002766:Cholecystokinin / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D010446:Peptide Fragments; D051381:Rats; D011919:Rats, Inbred Strains; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification / Q000378:metabolism*; D012844:Sincalide / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D002241:Carbohydrates; D006023:Glycoproteins; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; C053823:cholecystokinin (26-33), I-Tyr-Gly-Nle(28,31)-; D002766:Cholecystokinin; D006026:Glycoside Hydrolases; D012844:Sincalide