Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and was purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose and ligand affinity chromatography. The purified receptor displayed a single diffuse band with a Mr of 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment of the receptor with N-glycanase, removing N-linked oligosaccharide moieties, the protein yielded a Mr = 38,000 band. These results agree with the Mr value estimated for the GRP receptor that was labeled on Swiss 3T3 cells by cross-linking to 125I-GRP1-27. GRP1-27 bound to the purified receptor with a Kd of 0.038 +/- 0.019 nM. By comparison, the soluble receptor in unfractionated extracts and intact membranes displayed a Kd for GRP1-27 of 0.036 +/- 0.003 nM and 0.13 +/- 0.04 nM, respectively. The relative potencies of a series of GRP analogs for the soluble receptor and intact membranes indicated that the extraction procedure did not significantly alter the receptor's ligand binding specificity. However coupling of the receptor to its guanyl nucleotide regulatory protein was not maintained in the soluble extract, and a G-protein did not co-purify with the receptor. Physiological concentrations of NaCl greatly inhibited the binding of some GRP analogs to the receptor, while the binding of other analogs was not affected. A domain on the GRP molecule involving Lys-13 or Arg-17 was identified which promoted binding to the GRP receptor under conditions of low ionic strength. These findings aided the development of an effective ligand affinity resin for the purification of the GRP receptor.
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PMID:Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. 217 Mar 75

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.
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PMID:Identification of the bombesin receptor on murine and human cells by cross-linking experiments. 303 12

In the present study, we investigated the nature and the importance of glycosylation of two mammalian bombesin receptors, the neuromedin B receptor (NMB-R) and the gastrin-releasing peptide receptor (GRP-R), using chemical cross-linking and enzymatic deglycosylation. [125I]-(D-Tyr0)NMB cross-linked to native NMB-R on rat C-6 glioblastoma cells or rat NMB-R transfected into BALB 3T3 cells revealed a single broad band, M(r) = 63,000, on both cell types that was not altered by DTT. NMB inhibited cross-linking specifically and saturably with an IC50 of 4.8 and 6.1 nM for C-6 and NMB-R transfected cells, respectively, and there was a close correlation between its ability to inhibit binding and its ability to inhibit cross-linking. A single broad band of M(r) = 82,000 was cross-linked with [125I]GRP on mouse GRP-R transfected BALB 3T3 cells. Peptide-N4-(N-acetyl-beta- glucosaminyl)asparagine amidase F (PNGase F) digestion increased the mobility of the original band in C-6, NMB-R, and GRP-R transfected cell membranes. Endoglycosidase H (Endo-H) and endoglycosidase F2 (Endo-F2) digestion had no effect on both transfected cells. Neuraminidase digestion slightly increased the mobility of the original band in NMB-R transfected cell membranes; however, it had no effect on GRP-R transfected cell membranes. Endo-alpha-N-acetylglucosaminidase (O-glycanase) digestion subsequent to neuraminidase treatment showed no additional effect on either receptor. Serial partial deglycosylation of cross-linked NMB-Rs with PNGase F treatment for different incubation periods revealed one band of partially glycosylated receptor (53 kDa) besides the fully glycosylated and fully deglycosylated ones, showing that NMB-R has two oligosaccharide chains. Similarly, three partially deglycosylated species (72, 62, and 52 kDa) are seen with the GRP-R, indicating that the GRP-R has four oligosaccharide chains. Treatment of unlabeled membranes with PNGase F followed by affinity labeling resulted in fully deglycosylated NMB-R or 75% deglycosylated GRP-R. Deglycosylation of the NMB-R did not alter its affinity for NMB or alter G-protein coupling; however, 75% deglycosylation of the GRP-R both decreased its affinity for GRP and altered its ability to couple to G-proteins. The present results demonstrate that NMB-R on native and transfected cells is an N-linked sialoglycoprotein with two triantenary and/or tetraantenary complex oligosaccharide chains. The apparent M(r) of this sialoglycoprotein is 63,000, and this protein does not contain disulfide-linked subunits or O-linked carbohydrates.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Glycosylation of bombesin receptors: characterization, effect on binding, and G-protein coupling. 794 1