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 beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.
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PMID:All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase beta-subunit contain oligosaccharide. 283 26

Primary cultures of rat alveolar type II cells bind radiolabeled pulmonary surfactant protein A (SP-A) with high affinity. The binding of 125I-labeled SP-A is time- and temperature-dependent and is not accompanied by significant degradation. The binding process is saturable at low concentrations of SP-A (5 micrograms/ml), and unlabeled SP-A readily competes with labeled SP-A for cellular binding sites. Subsequent to binding, two pools of cell-associated 125I-labeled SP-A can be identified based upon sensitivity to trypsin at 0 degrees C. It is likely that the trypsin-sensitive pool comprises 125I-labeled SP-A bound to the cell surface and the trypsin-insensitive pool comprises the internalized protein. Scatchard analysis of cell surface binding of SP-A at 0.1-10 micrograms/ml shows positive cooperativity at concentrations between 0.1 and 1 micrograms/ml. Hill plots give nH = 1.34 +/- 0.08 with an apparent dissociation constant K'd = 1.02 +/- 0.32 micrograms/ml (which is 0.64 +/- 0.19 nM if the native molecular mass of oligomeric SP-A is assumed to be 1.6 MDa). The binding of SP-A to type II cells shows an absolute requirement for Ca2+. The putative receptor for SP-A is unaffected by treatment of type II cells with a variety of proteases and N-Glycanase (EC 3.5.1.52). Alveolar macrophages also exhibit high-affinity binding of SP-A, but rat lung fibroblasts and the alveolar epithelial cell line L2 exhibit only nonspecific binding.
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PMID:Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A. 284 Jun 67

The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glycoprotein as a constituent of purified gamma-aminobutyric acid/benzodiazepine receptor complex: structures and physiological roles of its carbohydrate chain. 303 54

The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.
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PMID:Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor. 305 9

A sialoglycoprotein, an integral component of the head plasma membrane of human spermatozoa, is recognized by the a-HS 1A.1 monoclonal antibody. The antigenicity is associated with the sugar moiety since: a) trypsin digestion did not affect the antigenic determinant; b) pretreatment of the cells with beta-glucosidase, alpha-mannosidase and neuraminidase completely abolished antibody binding. Endoglycosidase D and glycopeptidase F were inactive. The a-HS 1A.1 did not recognize a variety of blood-group related synthetic oligosaccharides. The species specificity was studied by indirect immunofluorescence assay. The antibody also recognized an antigen on Macaca fascicularis sperm, but failed to bind to spermatozoa of boar, bull, goat, ram, stallion, dog, rabbit, rooster, carp and eel.
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PMID:Primate specific sialoglycoprotein of sperm head plasma membrane defined by an anti-carbohydrate monoclonal antibody. 331 19

The biosynthesis and maturation of human sucrase-isomaltase (SI, EC 3.2.1.48-10), was studied in cultured small intestinal biopsy specimens and mucosa explants. Pulse-chase experiments with [35S]methionine revealed one high mannose intermediate of Mr = 210,000 (pro-SIh) which was processed at a slow rate to an endo H-resistant, mature form of Mr = 245,000 (pro-SIc). The fully core-glycosylated form (Mr = 212,000) was detected only when 1-deoxynojirimycin was added to the culture medium, thus indicating that the core sugars undergo rapid processing by rough endoplasmic reticulum membrane-bound glycosidases. The data presented showed that trypsin specifically and instantaneously (within 1 min) cleaves pro-SIc to two subunits Ic (Mr = 145,000) and Sc (Mr = 130,000). Elastase and chymotrypsin are not effective. Enzymic and chemical deglycosylations of SI with endo-beta-N-acetylglucosaminidase F/glycopeptidase F and trifluoromethanesulfonic acid (TFMS) as well as probing for the binding capacity of SI to Helix pomatia lectin demonstrated that pro-SIc, Ic, and Sc are N- and O-glycosylated. Furthermore, the results were indicative of a posttranslational O-glycosylation of pro-SI, since (i) the earliest detectable precursor form, pro-SIh, did not bind to H. pomatia lectin and (ii) its deglycosylation products with both endo-beta-N-acetylglucosamidase H and TFMS were identical. Both the Sc and Ic subunits contain eight N-linked glycan units, at least one of which is of the high mannose type and found on Sc. Finally, Sc, but not Ic, was shown to display at least four populations varying in their content of O-linked glycans. The heterogeneous O-glycosylation pattern of Sc could be correlated with the distal position of this subunit (and its O-glycosylation sites) within the pro-SI molecule, thus affecting the extent of O-linked oligosaccharide processing and their subsequent presentation on the mature molecule.
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PMID:Biosynthesis of the human sucrase-isomaltase complex. Differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex. 336 77

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

IgE-binding factors (IgE-BFs) were purified from the culture supernatant of RPMI-8866 cells, a human lymphoblastoid B-cell line expressing IgE receptors. The material, purified by affinity-chromatography on immunoadsorbents coupled to IgE or to monoclonal antibody against IgE receptor, was comprised of two major components with apparent molecular weight (MW) of 25,000-27,000 and 12,000, as determined by SDS-PAGE and silver staining. Only the 25,000-27,000 MW molecules were identified as IgE-BFs, as demonstrated by their reactivity with MabER in the Western blot and the immunoprecipitation assays, and their ability to inhibit rosette formation of U937 cells with IgE- but not with IgG-coated erythrocytes. IgE-BFs were purified to homogeneity by combining affinity-chromatography and either DEAE-ion exchange or reverse-phase chromatography on an HPLC system. Chromatofocusing analysis demonstrated the microheterogeneity of IgE-BFs that were comprised of molecules with isoelectric points ranging from 5.0 to 4.4. IgE-BFs were sensitive to treatment with O-glycosidase but not with N-glycanase. These molecules were resistant to heat and to pH ranging from 2 to 9; their immunoreactivity was lost after treatment with trypsin and pepsin. Papain digestion of purified IgE-BFs generated 14,000-16,000 MW molecules that were still binding to IgE and to MabER.
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PMID:Purification and partial biochemical characterization of IgE-binding factors secreted by a human B lymphoblastoid cell line. 349 83

Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation
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PMID:Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate. 353 42

The glycoprotein bovine fetuin was treated with trypsin and the Asn-81 tryptic glycopeptide was purified (90% pure by Edman sequencing) by reversed-phase chromatography (RP-HPLC). The Asn-81 glycopeptide, which eluted as a single peak by RP-HPLC, was separable into five peaks on the NucleoPac PA100 column, a pellicular anion-exchange column. Each of the five Asn-81 glycopeptide peaks was shown to contain N-linked oligosaccharides by treatment of each peak with peptide N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) and subsequent oligosaccharide analysis by high-pH anion-exchange chromatography with pulsed amperometric detection. High-pH anion-exchange chromatography-pulsed amperometric detection oligosaccharide analysis revealed that each peak contained a different population of sialylated N-linked oligosaccharides. Hence each peak contained a different group of glycopeptide glycoforms. It was observed that the longer the retention time of the Asn-81 glycopeptide peak on the anion-exchange column, the greater the oligosaccharide sialylation. Two glycopeptide peaks which differed in their distribution of disialylated oligosaccharides demonstrated that the glycopeptide separation was a result of something more than gross differences in sialic acid content. The two other N-linked tryptic glycopeptides of fetuin were also separated into multiple peaks on the NucleoPac PA100 column and these separations were shown to be due to differences in oligosaccharide sialylation. The separations of the three fetuin N-linked glycopeptides demonstrate that pellicular anion-exchange chromatography offers improved separation speed and resolution for the separation of sialylated glycopeptides.
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PMID:Improved fractionation of sialylated glycopeptides by pellicular anion-exchange chromatography. 751 57


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