Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 aim of this study was to isolate the Na(+)-independent bile acid transporter from rat canalicular plasma membranes by affinity chromatography. The affinity matrix used consisted of lysylcholic acid covalently linked to CH-Sepharose 4B, resulting in an anionic ligand essentially identical to glycocholic acid. The protein fraction, adsorbed and eluted from the affinity column, was markedly enriched in a 100-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared with the total membrane and membrane extract. The 100-kDa band, further purified by preparative SDS-PAGE, was electroeluted from excised gel fragments and used as an immunogen for antibody production in rabbits. The immune serum, but not preimmune serum, specifically recognized a single, 100-kDa polypeptide on one- and two-dimensional immunoblots of canalicular membranes. In contrast, no reactivity was observed with proteins in liver basolateral or ileal brush-border membranes. The 125I-labeled protein was immunoprecipitated from membrane extracts solubilized in NP-40 and was found to migrate with a pI of 5.3 on two-dimensional electrophoresis. The apparent molecular weight of the protein was reduced by 50% after deglycosylation with N-glycanase. The 100-kDa protein was localized specifically and exclusively by immunocytochemical methods to the bile canalicular domain of the hepatocyte plasma membrane. Moreover, the immunoglobin G fraction prepared from the antiserum significantly inhibited taurocholate transport by canalicular membrane vesicles and decreased the covalent labeling of the 100-kDa protein by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus the isolation of a single 100-kDa protein by bile acid-affinity chromatography, as well as the inhibitory effects of antibodies directed against this polypeptide, provide further support for its role in the canalicular transport of bile acids.
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PMID:Isolation and characterization of the canalicular membrane bile acid transport protein of rat liver. 218 68

N-Glycosidase F (peptide-N4-(N-acetyl-beta-glycosaminyl)asparagine amidase; EC 3.5.1.52) catalyzes the cleavage of N-glycosidically linked carbohydrate chains between N-acetylglucosamine and asparagine. The structural gene was isolated by screening a Flavobacterium meningosepticum genomic DNA library in lambda gt10 with oligonucleotides, deduced from partial amino acid sequences of the protein. A clone with an open reading frame of 1062 bases was obtained. The amino acid sequence reveals a 42-residue-long leader peptide, which shows similarities to the endoglycosidase H-leader with respect to the cleavage site of the signal peptide, but is distinct from the ones known from other Gram-positive or -negative bacteria. The molecular weight of the native protein, derived from the DNA sequence, is in agreement with the molecular weight of the purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (35,000). Escherichia coli, transformed with a plasmid containing this DNA sequence, expresses N-glycosidase F activity. The enzyme with its natural Flavobacterium promoter and leader peptide is not secreted in E. coli but seems to be associated with cell membranes.
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PMID:Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum. 220 81

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.
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PMID:Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells. 235 Apr 87

Neuroglandular antigen (NGA) was identified as a human melanoma-associated antigen by a panel of murine monoclonal antibodies of both IgG2a (LS62, LS76, LS159) and IgG1 (LS113, LS140, LS152) subclasses, developed in this laboratory (L. Sikora, A. Pinto, D. Demetrick, W. Dixon, S. Urbanski, and L. M. Jerry, Int. J. Cancer, 39: 138-145, 1987). Monoclonal antibody LS62 was used to immunoprecipitate NGA from radiolabeled cultured melanoma cells, and it behaved as a heterogeneous glycoprotein "smear" on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (Mr 29,000-70,000). Radioactive pulse-chase time course experiments using human melanoma cells cultured in the presence or absence of inhibitors of protein glycosylation showed that the antigen consisted of a core protein with a molecular weight of 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule was modified by the addition of at least three N-linked oligosaccharide side chains (as revealed by limited N-glycanase digestion) to give a precursor form with a molecular weight of approximately 34,000. Subsequent processing steps yielded a heterogeneous family of glycoproteins with varying amounts of covalently attached carbohydrate. Much of this heterogeneity in both molecular weight and pI (as revealed by two-dimensional electrophoresis) could be removed by treatment of the antigen with neuraminidase, suggesting heavy sialylation of the glycoprotein. NGA could be detected on the surface of melanoma cells by fluorescence-activated cell sorter analysis, surface radioiodination, and, as previously shown, immunoperoxidase staining. However, there was a larger intracellular pool of the molecule and the antigen was rapidly released into the culture supernatant. The function of NGA remains unknown but its elevated expression in transformed melanocytes have prompted this characterization to understand its biochemical nature and relation to other melanoma-associated antigens.
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PMID:Biosynthesis, glycosylation and intracellular processing of the neuroglandular antigen, a human melanoma-associated antigen. 236 31

Porcine 32,000 Mr inhibin is a glycoprotein with one asparagine-linked glycosylation site on the alpha-subunit. The presence of carbohydrate on the alpha-subunit was visualized by periodate-Schiff (PAS) staining. This stain for carbohydrate also verified that the beta-subunit of 32,000 Mr porcine inhibin does not contain carbohydrate. When analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) under reducing conditions, the inhibin alpha-subunit consistently existed as a doublet, and by the PAS stain, both bands of the doublet were glycosylated. Analysis by two-dimensional (2D) PAGE further revealed the presence of charge isoforms of the alpha-subunit. The alpha-subunit of inhibin could be deglycosylated by N-glycanase, but not by endoglycosidase F, endoglycosidase D, or endoglycosidase H. When the N-glycanase-treated inhibin was analyzed by either 1D-PAGE or 2D-PAGE, the molecular size of the alpha-subunit was reduced by 3500 Mr. Each doublet band observed with reducing conditions in 1D-PAGE or 2D-PAGE for the alpha-subunit became a single band (spot) in the deglycosylated alpha-subunit. However, the charge heterogeneity detected by 2D-PAGE was retained, indicating that only a portion of this heterogeneity is attributable to the carbohydrate moiety. The in vitro biological activity of the deglycosylated inhibin was not different from the control sample. The composition of the carbohydrate in inhibin was investigated with the Dionex carbohydrate analyzer. Inhibin contains fucose, glucosamine, galactose, mannose, and glucose. Colorimetric analysis revealed the presence of sialic acid. Taken together, this implies some aspect of the peptide portion of the molecule is involved in charge heterogeneity. Inhibin may have an unusual carbohydrate component, as evidenced by the detection of glucose in inhibin samples. The absence of glucose in the carbohydrate moiety of another glycoprotein fraction that accompanied the inhibin through all the same fractionation procedures argues against the artifactual introduction of glucose in the fractionation medium per se.
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PMID:The heterogeneity of porcine 32,000 Mr inhibin alpha-subunit: a gel electrophoresis and immunoblot study. 238 62

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.
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PMID:Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation. 240 51

A variety of homobifunctional crosslinking agents have been used to gain insight into the nature of the murine interleukin 3 (mIL-3) receptor. When [125I]mIL-3 was cross-linked to receptor sites on the surfaces of intact B6SUtA1 cells with disuccinimidyl suberate (DSS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two radiolabeled species with molecular weights of 140 (p140) and 70 (p70) kd (after subtraction of [125I]mIL-3). The relative intensities of the two bands did not change when the [125I]mIL-3 concentration was varied, confirming Scatchard results which suggested only one affinity class. However, when [125I]mIL-3 was crosslinked to intact cells and then incubated at 37 degrees C, the intensity of p140 decreased relative to p70, suggesting a conversion of p140 to p70. This conversion could be inhibited by sodium azide, methylamine, and bacitracin and could also be prevented by first boiling for 1 min in 2% SDS and 5% 2-mercaptoethanol. The putative protease that carried out this apparent conversion appeared to be associated both with plasma membranes prepared from these cells and also with solubilized receptors. Moreover, when p140, crosslinked with both dithiobis succinimidylpropionate and glutaraldehyde, was purified and reelectrophoresed under reducing conditions, p70 could be generated. N-glycanase digestion of p140 and p70 revealed a similar level of N-linked carbohydrate, which upon closer study appeared to consist of two chains, a 3-kd and an 8-kd moiety. Consistent with this data, we propose that the receptor is a 140-kd glycoprotein that is cleaved to a 70-kd surface protein upon mIL-3 binding and chemical crosslinking.
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PMID:Characterization of the interleukin 3 receptor. 240 77

Although antigen-reactive T lymphocytes play a central role in the host response to Histoplasma capsulatum, little is known of the nature of Histoplasma antigens recognized by these cells in vitro. Employing a murine T-cell line and two clones that are reactive with histoplasmin, we examined whether activation of T cells by histoplasmin required the presence of carbohydrate or protein moieties. The approach taken was to modify carbohydrate or protein molecules in histoplasmin by chemical or enzymatic digestion or by lectin adsorption. In parallel, antigen was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate alterations in functional activity with changes in the electrophoretic appearance of histoplasmin. Treatment of histoplasmin with periodate (0.1 M, 0.05 M, and 0.01 M) or with the endoglycosidases N-glycanase and endoglycosidase H sharply diminished the capacity of histoplasmin to trigger responses by T cells. Reactivity of T cells to histoplasmin that had been adsorbed with lectins binding mannose, glucose, or galactose was reduced by greater than 70%; conversely, the responses by T cells to antigen that had been adsorbed with lectins specific for fucose, N-acetylgalactosamine, or N-acetylglucosamine ranged from 82 to 91% of that to control antigen. Proliferative responses by T cells to histoplasmin that had been digested with chymotrypsin, protease, or trypsin were 2 to 43% of control values. The electrophoretic appearance of histoplasmin was modified by some but not all of the treatments. Partially purified H and M antigens triggered proliferation of T cells. Thus, both carbohydrates and proteins must be present to induce optimal responses by T cells. A portion of the carbohydrates is N linked to proteins, and alpha-D-mannose (or alpha-D-glucose) and beta-D-galactose are the sugar ligands of carbohydrate-containing antigens.
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PMID:Characterization of antigenic determinants in histoplasmin that stimulate Histoplasma capsulatum-reactive T cells in vitro. 245 54

Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.
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PMID:Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component. 247 51


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