Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor.
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.
Pearson
R K
RK
Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Miller
L J
LJ
Hadac
E M
EM
Powers
S P
SP
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
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