Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor.

R. K. Pearson, Laurence J Miller, E. M. Hadac, S. P. Powers

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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.

Original languageEnglish (US)
Pages (from-to)13850-13856
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number28
StatePublished - Oct 5 1987

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Cholecystokinin Receptors
Cholecystokinin
Glycoproteins
Carbohydrates
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Digestion
Chemical analysis
Oligosaccharides
alpha-N-Acetylgalactosaminidase
Proteins
Sialoglycoproteins
Hydrofluoric Acid
Neuraminidase
Lectins
Proteolysis
Triticum
Chromatography
Carrier Proteins
Labeling
Rats

ASJC Scopus subject areas

  • Biochemistry

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Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor. / Pearson, R. K.; Miller, Laurence J; Hadac, E. M.; Powers, S. P.

In: Journal of Biological Chemistry, Vol. 262, No. 28, 05.10.1987, p. 13850-13856.

Research output: Contribution to journalArticle

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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.",
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