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 44G4 antigen is expressed in high amounts on human endothelial cells and at low levels on leukemic cells of pre-B and myelomonocytic origin. Its level of expression on the pre-B leukemic HOON cell line used for derivation of the corresponding mAb is intermediate but sufficient to permit the purification of the Ag. The molecule isolated by immunoaffinity from HOON is a glycoprotein since it bound to Ricinus communis agglutinin, wheat germ agglutinin, and peanut agglutinin lectins. The Ag was purified 2400-fold from a soluble taurocholate extract of HOON cells by affinity to wheat germ agglutinin-agarose and 44G4-IgG-Sepharose. The purified glycoprotein is likely a homodimer as it migrated on SDS-PAGE with an apparent m.w. of 170,000, nonreduced, and 95,000, reduced. Removal of N-linked oligosaccharides by endoglycosidase F led to a decrease in m.w. of 25,000; if neuraminidase and O-glycanase were also present, the total decrease in m.w. was 33,000 suggesting a polypeptide chain of 62,000 and 8,000 in O-linked substitutions. The glycoprotein digested with N-glycanase, neuraminidase, or O-glycanase could still be immunoprecipitated with the 44G4 mAb indicating that the antigenic epitope resides in the polypeptide. By Western blot analysis, the dissociated but nonreduced protein was reactive with 44G4, whereas the reduced and alkylated protein was not. Therefore, the epitope is dependent on the presence of intact disulfide bond(s). Sequential immunoprecipitation with OKT9 and 44G4 antibodies indicated that these epitopes are present on two distinct molecules and that 44G4 is distinct from the transferrin receptor despite a similar subunit structure.
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PMID:Biochemical characterization of the 44G4 antigen from the HOON pre-B leukemic cell line. 326 45

The p150,95 cell surface protein is a member of a family of heterodimeric leukocyte adhesion proteins that have homologous alpha subunits, each noncovalently associated with a common beta subunit. In this report we have metabolically labeled the U937 cell line at various timepoints during its phorbol myristic acetate-induced maturation to examine the kinetics of synthesis of these proteins during monocytic differentiation, and their maturation and glycosylation. The p150,95 alpha subunit was immunoprecipitated with p150,95-specific monoclonal antibody (MAb), or an antiserum to the denatured, purified alpha X subunit. The glycosylation and polypeptide chain length of the p150,95, Mac-1, and lymphocyte function associated antigen (LFA-1) alpha and beta subunits were compared by immunoprecipitation with subunit specific MAb and antisera, and by digestion with Endo H and N-glycanase. The p150,95 alpha subunit is synthesized as a precursor of 146,000 Mr, has five to six N-linked oligosaccharides, and has a polypeptide chain backbone of 132,000 Mr. Over 50% of the carbohydrate on the mature alpha subunit of 150,000 Mr was sensitive to Endo H digestion. The p150,95 alpha and beta precursors can associate before maturation into the mature form. Conversion to the mature form was accompanied by loss of reactivity with the antiserum to the denatured alpha X subunit, suggesting a change in conformation. Mac-1 and LFA-1 alpha subunits have precursors of 160,000 Mr and 165,000 Mr, respectively, and contain N-linked carbohydrates. The polypeptide chain length for the Mac-1 alpha subunit is 137,000 Mr, and for LFA-1 is 149,000 Mr. Only 14% of the oligosaccharide on the mature LFA-1 alpha subunit was sensitive to Endo H, suggesting that unlike p150,95, most is converted to the complex type. The differences noted in the Mr of the three homologous alpha subunits are therefore due to differences in both polypeptide chain length and carbohydrate processing during biosynthesis.
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PMID:Biosynthesis and glycosylation of p150,95 and related leukocyte adhesion proteins. 329 34

Thyroglobulin from colloid as well as from membrane fractions became radiolabeled upon incubation of calf thyroid slices with [35S]sulfate. The identity of the sulfate-labeled molecule was established by immunoprecipitation, polyacrylamide gel electrophoresis, Bio-Gel A-5m filtration, and DEAE-cellulose chromatography. Size analysis by gel filtration of [35S]glycopeptides and hydrazine-released oligosaccharides indicated that the sulfate was primarily located in the complex (unit B) carbohydrate units of thyroglobulin. Moreover, although [35S]sulfate-labeled oligosaccharides were cleaved by N-glycanase to the same extent as those labeled with [3H]mannose, they were not released by endo-beta-N-acetylglucosaminidase under conditions that led to the complete removal of polymannose carbohydrate (unit A). The failure of 35S-labeled glycopeptides and oligosaccharides to bind to immobilized Concanavalin-A indicated that the sulfate residues in calf thyroglobulin are located in carbohydrate units with three or more branches. No evidence for the occurrence of tyrosine sulfate was found upon examination of Pronase digests of radiolabeled thyroglobulin, and chemical analyses excluded the presence of this amino acid down to a level of 0.5 residues/polypeptide subunit. Studies with density gradient-separated membrane fractions as well as with puromycin indicated that sulfate addition is a late event in thyroglobulin biosynthesis which occurs in the Golgi compartment. Furthermore, it was observed that the nondimerized thyroglobulin subunit was much less sulfate labeled than the mature molecule. The location of the sulfated carbohydrate in a terminal portion of the calf thyroglobulin peptide chain was suggested by the observation that the subunit [mol wt (Mr) = 330,000] can undergo a transformation, presumably mediated by an endogenous protease, to a sulfate-free component (Mr = approximately 270,000) with the appearance of a 35S-labeled 60,000 Mr fragment; the release of a single sulfate-labeled peptide (Mr = 60,000) by mild trypsin treatment was consistent with a sequestration of sulfate groups in the thyroglobulin molecule.
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PMID:Biosynthesis of sulfated asparagine-linked complex carbohydrate units of calf thyroglobulin. 338 87

Antiserum was raised in rabbits against a bile canalicular glycoprotein of Mr = 110,000 purified to homogeneity from of rat liver. The antisera specifically immunoprecipitated a Mr = 110,000 polypeptide from hepatocytes metabolically labeled with [35S]methionine. When hepatocytes in primary culture were incubated with tunicamycin before labeling with [35S]methionine in the presence of tunicamycin, the major polypeptide immunoprecipitated by the specific antiserum from Triton X-100 extracts of cells had a molecular weight of 59,000. Enzymatic removal of N-linked carbohydrates from the Mr = 110,000 glycoprotein by N-glycanase digestion also yielded a polypeptide with minimum Mr = 59,000. In pulse-chase experiments using [35S]methionine, the Mr = 110,000 protein detected by the specific antisera first appears as Mr = 85,000 and 75,000 intermediate species which are endoglycosidase H sensitive. The Mr = 85,000 intermediate form is lost first with time followed by the Mr = 75,000 form giving rise to the Mr = 110,000 form that is endoglycosidase H resistant. Neuraminidase digestion of the Mr = 110,000 form generated an Mr 85,000 form but with a different carbohydrate structure than the intermediate Mr 85,000 form detected in the pulse-chase experiments. The time required to accomplish the processing of the Mr = 85,000 and 75,000 forms is relatively slow. Finally, the terminal sugars are added and the mature Mr = 110,000 glycoprotein is rapidly transported to the cell surface. A minimum time of 90 min is required for the Mr = 110,000 bile canalicular glycoprotein to be synthesized, processed, and reach the cell surface which is long relative to the time required (10 min) for another domain-specific protein, the receptor for asialoglycoproteins, to reach the sinusoidal surface. The Mr = 110,000 bile canalicular glycoprotein turns over in the bile canalicular domain with a half-life of 43 h while the asialoglycoprotein receptor turns over in the sinusoidal domain with a half-life of 23 h.
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PMID:Biosynthesis and turnover of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus. 366

The possible noninvolvement of the carbohydrate moiety of human fibrinogen in the clotting mechanism was examined by eliminating the neutral sugar chains from desialylated fibrinogen by almond glycopeptidase digestion. 40% of the total neutral sugars was removed from the desialylated fibrinogen. The neutral sugars from both the beta- and gamma-polypeptide chains were released equally. The protein moiety of the glycopeptidase-digested fibrinogen was found to be intact. No significant change was observed in the thrombin time(fibrinogen clottability) of the resultant fibrinogen. The results suggest that the carbohydrate moiety of fibrinogen is not involved in the clotting mechanism. Oligosaccharide was detected in the glyopeptidase digest of desialylated fibrinogen by thin-layer chromatography (TLC), and was found to be identical with those released quantitatively from the peptic digests of beta- and gamma-polypeptide chains. The structure of the sugar chain was identified tentatively as Gal2-GlcNAc2-Man3-GlcNAc2, by sequential exoglycosidase digestion and quantitative analysis of carbohydrate components.
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PMID:The release of carbohydrate moieties from human fibrinogen by almond glycopeptidase without alteration in fibrinogen clottability. 729 38

Various alpha and beta 3 subunit-specific antibodies were used to characterize some of the heterogeneous ligand-binding properties of gamma-aminobutyric acidA receptors. Polyclonal antibodies that were raised against the cytoplasmic amino acid sequence (380-392) of the rat beta 3 subunit recognized a single polypeptide of molecular mass of 58 kDa in Western blots with Ro7-1986 affinity-purified GABAA receptors from the rat brain, and a doublet of molecular mass of 54 kDa and 56 kDa in receptors from the bovine cortex, hippocampus, and cerebellum. Deglycosylation of purified receptors from the bovine cortex with N-glycanase resulted in a single band immunostained at molecular mass of 52 kDa. These anti-beta 3 subunit antibodies immunoprecipitated approximately 50% of [3H]flunitrazepam binding sites from soluble extracts of bovine cortex, whereas beta cyto antibodies, which probably recognize all beta subunit isoforms, precipitated almost 100% of benzodiazepine binding sites. These results indicate heterogeneity of GABAA receptor subunit composition with respect to the nature of beta subunits. The GABA analogue 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), like GABA, shows heterogeneous binding affinities in brain homogenates. The higher affinity sites were previously suggested as corresponding to a 58-kDa polypeptide in rat that is photoaffinity-labeled with [3H]muscimol, a band that comigrates with the one stained by anti-beta 3 antibodies. However, THIP affinity was not significantly different between receptors containing beta 3 subunits and those lacking beta 3, as demonstrated by similar affinities in receptors that ere immunoprecipitated by anti-beta 3 antibodies and those that were not. Also, THIP displaced [3H]muscimol binding with similar multiple affinities across brain regions where different beta subunit variants are expressed with varying abundances. These observations suggest that the property of high affinity THIP binding cannot be explained solely by beta 3 subunits. The coupling efficiency between GABA and benzodiazepine binding sites appears to be determined by the nature of alpha subunits rather than of beta subunits. GABA enhanced [3H]flunitrazepam binding with different efficacies and potencies in receptors immunoprecipitated by anti-alpha 1, -alpha 2, and -alpha 3 subunit antibodies. In contrast, beta 3 subunit-enriched and disenriched receptors did not differ in this property. [3H]Flunitrazepam binding in GABAA receptors containing alpha 2 and alpha 3 subunits was enhanced to a significantly greater extent than were those with alpha 1. In addition, receptors containing alpha 1 and alpha 3 subunits had higher potencies of enhancement than did those with alpha 2 subunits.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pharmacological subtypes of the gamma-aminobutyric acidA receptors defined by a gamma-aminobutyric acid analogue 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol and allosteric coupling: characterization using subunit-specific antibodies. 747 92

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").
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PMID:Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin. 763 98

Over-expression of a 95-kDa membrane protein (P-95) has been reported previously in the multi-drug-resistant (MDR) breast cancer cell line MCF-7/AdrVp and the MDR small cell lung cancer line NCI-H1688. We have now developed anti-sera against gel-purified }-95 protein from each of these cell lines. Western blotting with each serum demonstrates a broad band at 95-kDa with detergent-solubilized membrane proteins from MCF-7/AdrVp and NCI-H1688 cells, which is barely detectable in membrane proteins from drug-sensitive parental MCF-7 cells. Each anti-serum cross-reacts with a 190-kDa membrane protein (P-190) in NCI-H1688 but not MCF-7/AdrVp cells. Immunoblotting and silver staining of NCI-H1688 membrane proteins separated by two-dimensional gel electrophoresis demonstrates that P-95 and P-190 run as broad streaks with low iso-electric points. Incubation of NCI-H1688 cells with tunicamycin or cleavage of carbohydrate residues of NCI-H1688 or MCF-7/AdrVp membrane proteins with PNGase F leads to the appearance of a sharp 35-kDa band reactive with anti-P-95 antisera. This 35-kDa protein has been isolated by two-dimensional gel electrophoresis. Neuraminidase digestion converts P-95 to a broad 65-kDa immunoreactive band, indicating the presence of terminal sialic acid residues. In conclusion, P-95 is an N-linked sialoglycoprotein with a 35-kDa polypeptide core.
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PMID:Characterization of a 95 kilodalton membrane glycoprotein associated with multi-drug resistance. 766 31

The carbohydrate moiety of the envelope glycoprotein gp51 of bovine leukemia virus, American strain, was studied. The virus was grown in ovine, bovine, porcine, bat and rat cells of various organ specificities. The gp51 was purified by immunoaffinity chromatography from virions of ten different virus-producing cells derived from various body organs of different species. Highly purified glycoproteins (single band in PAGE) were compared for their electrophoretic mobility, for the presence of epitopes by a battery of monoclonal antibodies, and for the glycosylation pattern by lectin blot analysis. Electrophoretic analysis of all tested glycoproteins deglycosylated by glycopeptidase F detected the same polypeptide backbone according to PAGE. The glycoproteins produced in rat cells migrated faster in PAGE, as detected in cells or in virions, than those produced in ovine cells. The pattern of their glycosylation was found to be dependent on the type of cells used for virus production. The differences in glycosylation were most pronounced when comparing the glycoprotein produced in ovine cells versus bat or rat cells. Changes in epitope expression were also detected. The differences in the patterns of glycosylation and in the accessibility of epitopes owing to the virus production in various kind of cells are discussed from virus infectivity and vaccine points of view.
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PMID:Envelope glycoprotein gp51 of bovine leukemia virus is differently glycosylated in cells of various species and organ origin. 768 44

The dopamine transporter (DAT) in rat striatum was examined during postnatal development and aging after photolabeling with [125I]DEEP. The DAT-[125I]DEEP protein complex from adult rats (2 months) appeared as a broad diffuse band in SDS-PAGE gels with average apparent molecular mass of about 80,000 Da as previously found. However, the molecular mass was lower at birth (day 0) and at postnatal ages 4 and 14 days. In aged rats (104 weeks), the molecular mass was slightly higher than that found in young adults (60 days). In binding experiments with [3H]BTCP, there were age-related differences in Kd and Bmax with decreases in both Kd and Bmax found in aged rats. Treatment of photolabeled membranes with neuraminidase caused a reduction in DAT molecular mass, but age-related differences were maintained. Treatment with N-glycanase greatly reduced or eliminated the age-related differences. Several DAT peptide-specific polyclonal antibodies immunoprecipitated DAT-[125I]DEEP protein complex at different developmental ages. Taken together, these results suggest differential glycosylation of rat DAT occurs during postnatal development and aging; the increase is due to increases in the N-linked sugars rather than changes in either sialic acid content or the polypeptide.
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PMID:Developmentally regulated glycosylation of dopamine transporter. 769 70


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