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)

Affinity labeling using [125I-Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both M(r) = 50,000 to 60,000 and M(r) = 96,000 to 115,000 [1,2]. In this work the glycoprotein nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N-glycosidase F (PNGase F), and O-glycanase treatment. Only N-glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16-17 kDa units, that is M(r) = 96,000 to 79,000 and M(r) = 60,000 to 44,000, indicating removal of N-linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N-linked complex (sialo)glycoproteins with a minimal core protein size of M(r) = 44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn-linked carbohydrate may be of the tri- or tetra-antennary complex type containing terminal sialic acid residues.
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PMID:Y2 receptor proteins for peptide YY and neuropeptide Y. Characterization as N-linked complex glycoproteins. 132 32

Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo-beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N-glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56-53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor.
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PMID:Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins. 171 81

We have used a cloned fusion protein as antigen to generate an antiserum specific for the rat Na,K-ATPase beta 2 subunit. Utilizing this antiserum, we analyzed some of the structural features and tissue distribution of the beta 2 subunit. Treatment of a rat brain microsomal membrane fraction with N-glycanase F revealed that the beta 2 subunit is composed of an approximately 32 kDa core protein and at least two N-linked carbohydrate chains. The beta 2 subunit also was found to copurify with ouabain-inhibitable Na,K-ATPase activity from rat brain. Western blot analysis of rat tissue microsomes showed that beta 2 subunits were expressed in brain, pineal gland, and thymus. However, no beta 2 subunits were detected in kidney, heart, spleen, liver, mammary gland, or lung. These results suggest that the beta 2 subunit is a functional component of the rat brain Na,K-ATPase. The restricted tissue distribution of beta 2 subunits may reflect important differences in the functions of individual beta subunit isoforms.
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PMID:The Na,K-ATPase beta 2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity. 215 63

The cytoplasmic tubulovesicular and canalicular membranes of gastric parietal cells are intimately involved in hydrochloric acid secretion. To characterise the glycoproteins of these membranes, we examined a panel of lectins for reactivity with parietal cells in paraffin sections of rat, dog and pig stomach. The poly-N-acetyllactosamine-specific lectin from Lycopersicon esculentum (tomato) and from Solanum tuberosum (potato), and the galactose-specific lectin Ricinus communis agglutinin (RCA120), showed strong cytoplasmic binding of parietal cells of all three species, with a pattern indicative of an intracellular membrane network. Binding to parietal cells was confirmed by double-labelling studies with parietal cell auto-antibodies from patients with autoimmune gastritis. Mucous cells and mucin also bound these lectins strongly. Other gastric cell types did not stain with either tomato or potato lectin, but stained weakly with RCA120. Electron-microscopic examination of lectin binding sites using biotinylated tomato lectin or RCA120 and streptavidin-gold, revealed specific binding to the luminal face of parietal cell tubulovesicular and canalicular membranes as well as the contents of mucous cell secretory granules. Tomato lectin and RCA120 reacted by lectin blotting with a major species of apparent molecular weight 60-90 X 10(3) Mr from rat, dog and pig gastric membranes. A tubulovesicular membrane fraction, enriched 10-fold for K(+)-dependent phosphatase activity, was also enriched three-fold for tomato lectin binding as assessed by a solid-phase lectin assay. The 60-90K (K = 10(3) Mr) component, in 125I-labelled detergent extracts of dog tubulovesicular membranes, bound to an affinity support of tomato lectin-Sepharose and was specifically eluted with N,N',N'-triacetylchitotriose. Digestion with N-glycanase collapsed the 60-90K component into a sharp 35K band. We conclude that: (1) a 60-90K membrane glycoprotein localised on the luminal face of tubulovesicles and canaliculi of parietal cells interacts strongly with tomato lectin and RCA120; and (2) the glycoprotein is composed of a 35K core protein glycosylated with N-glycans probably containing poly-N-acetyllactosamine sequences with terminal galactosyl residues. The properties of this 60-90K glycoprotein are identical to a major parietal cell autoantigen recognised by sera of patients with autoimmune gastritis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Poly-N-acetyllactosamine-specific tomato lectin interacts with gastric parietal cells. Identification of a tomato-lectin binding 60-90 X 10(3) Mr membrane glycoprotein of tubulovesicles. 216 41

The monoclonal antibody (MAb) 5T4 defines a human trophoblast antigen marker with a restricted pattern of expression in normal adult tissues but this antigen is expressed on a variety of carcinomas. The purification of 5T4 antigenic molecules is described from term syncytiotrophoblast by a combination of lectin- and immunoaffinity chromatography and gel filtration giving up to 10,000-fold purification with 70% yield. The antigen is carried by non-associated glycoprotein molecules with an apparent molecular weight of 72 kDa on SDS-PAGE and a neutral pI. Removal of N-linked sugars by N-glycanase reveals a core protein of 42 kDa. Treatment with enzymes that cleave O-linked sugars does not substantially alter the molecular size. The native 5T4 molecules are very resistant to proteolysis until the N-linked sugars are removed or the glycoprotein is denatured and reduced. Glycopeptides generated by these approaches will be suitable for amino acid sequencing.
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PMID:Isolation and characterization of 5T4, a tumour-associated antigen. 229 3

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

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.
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PMID:The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae). 240 Dec 94

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).
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PMID:Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody. 244 76

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

The low molecular weight proteoglycan fraction extracted from articular discs with 4 M guanidinium chloride was found to consist predominantly of an iduronate-rich dermatan sulphate proteoglycan, together with chondroitin sulphate-containing material. The dermatan sulphate proteoglycan was purified by ion-exchange and gel-filtration chromatography and its core protein isolated after digestion with chondroitinase ABC. The amino acid composition and pattern of cyanogen bromide peptides obtained from this core were closely similar to those of the protein core of bovine skin proteodermatan sulphate. Four monoclonal antibodies raised against bovine skin proteodermatan sulphate also reacted with the disc protein core and its cyanogen bromide peptides. Results of digestion with glycopeptidase F demonstrated the presence of three N-linked oligosaccharides. The combined size of these oligosaccharides appeared to be somewhat less than the size of those on skin proteodermatan sulphate. The glycosaminoglycan chain released by digestion with cathepsin C had a higher molecular weight than that from skin. These differences in glycosylated structures may be responsible for the different effects on collagen fibrillogenesis in vitro; whereas skin proteodermatan sulphate only reduced the rate of fibril growth, disc dermatan sulphate proteoglycan also increased the length of the lag-phase and the final opacity.
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PMID:Proteoglycans of the articular disc of the bovine temporomandibular joint. II. Low molecular weight dermatan sulphate proteoglycan. 279 47


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