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 essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.
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PMID:Expression of blood clotting factor VIII:C gene in capillary endothelial cells. 162 40

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.
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PMID:Human tumour cathepsin B. Comparison with normal liver cathepsin B. 163 35

GABAA receptors purified from the brains of 5- to 10-day-old rats were completely N- and O-deglycosylated using N-glycanase and/or neuraminidase plus O-glycanase. Intact or completely deglycosylated receptors were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis. Polyclonal antibodies directed against synthetic aminoacid sequences specific for the GABAA receptor alpha 1-, alpha 2- or alpha 3-subunits each identified an apparently single protein of about 51 kDa, 53 kDa or 59 kDa, respectively, in the intact receptors. In the deglycosylated receptors, however, three different proteins were identified by antibodies directed against the alpha 3-subunit and at least two different proteins were identified by antibodies directed against the alpha 2- or alpha 1-subunit.
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PMID:Identification of alpha 1-, alpha 2- and alpha 3-subunit isoforms of the GABAA-benzodiazepine receptor in the rat brain. 166 May 79

Immunochemical methods have been used to detect and characterize two classes of polypeptide-associated keratan sulphate (KS) in epithelial secretions from human endometrium. Monoclonal antibody D9B1 binds to a hormonally regulated sialylated epitope associated with KS in a high relative molecular mass (250,000-350,000) component that bands as a doublet in SDS/PAGE. These KS chain(s) are sensitive to keratanase, endo-beta-galactosidase and N-glycanase. A second, more highly sulphated, type of KS is also present, that is resistant to all three enzymes. This can be detected using monoclonal antibody 5D4. It is present throughout the menstrual cycle and is associated principally with a component of Mr 140,000. Thus secretory KS contributes to the environment of the implanting embryo, may be used as a molecular index of endometrial function and could be important in the establishment of pregnancy.
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PMID:Menstrual-cycle-dependent expression of keratan sulphate in human endometrium. 169 31

A polyclonal antiserum was raised to a gel purified preparation of the major water-soluble surface glycoprotein (gp29) of adult Brugia malayi, and used to define the stage specificity of expression, localisation (by immunoelectron microscopy) and the dynamics of biosynthesis and turnover via pulse-chase experiments. Gp29 was not detected in surface-labelled preparations of either pre- or post-parasitic third stage larvae (L3), but was present in fourth stage larvae (L4), where its mass was estimated to be 30 kDa by SDS-PAGE. In both L4 and adult worms, the protein resolved as 3 distinct species in 2-dimensional electrophoresis, with pIs from 6.5 to 7.5. Pulse-chase studies via metabolic labelling of adult worms with [35S]methionine in vitro indicated that gp29 was processed from a 32-kDa precursor to the mature molecule within 45 min and that it was secreted into culture medium within 5 h of synthesis. On extended culture, gp29 was converted to a 56-kDa product, presumably either by complex formation or covalent linkage with another secreted molecule. This higher molecular weight component had a more acidic pI of 4.5 and was insensitive to digestion with N-glycanase. Immunoelectron microscopy showed that gp29 was distributed throughout the cuticle and hypodermal cell layer of adult worms, suggesting that the protein was synthesised in the hypodermis, and that turnover into culture medium occurred through the cuticle. The protein appeared to concentrate at the distal cell membrane of the hypodermis, particularly at the stacked invaginations. Additional immunostaining was found on the basement membrane of the basal lamina of the intestine.
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PMID:Cuticular localisation and turnover of the major surface glycoprotein (gp29) of adult Brugia malayi. 170 Feb 98

The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.
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PMID:Studies on the biochemical structure of the major cat allergen Felis domesticus I. 171 68

Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded.
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PMID:Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation. 172 Mar 95

The 18 kDa and 32 kDa lectin binding proteins of Chlamydia trachomatis were characterized as glycoproteins by treatments with glycosidases. The proteins of the serovar L2 whole cell lysate were separated by SDS-PAGE and transferred to nitrocellulose paper. After treatment with an enzyme, the proteins were reacted with a biotinylated lectin. Each of the endoglycosidases tested affected the binding of the lectin to the protein. PNGase F inhibited the binding of Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin I (UEAI) to both the 18 kDa and 32 kDa proteins. Endoglycosidase F and H inhibited the binding of these lectins to the 32 kDa protein completely and to the 18 kDa protein partially. In the exoglycosidase treatments, alpha-L-fucosidase prevented binding of only UEAI to the two proteins while beta-galactosidase inhibited the binding of SBA. Mannosidase abolished the binding of all the lectins tested. Neuraminidase had no effect. The proteins isolated by electroelution from the excised gels after SDS-PAGE were digested with an endoglycosidase. PNGase F-treated proteins showed a lower molecular weight mobility in which the lectin binding ability was destroyed. Endo-alpha-N-acetylgalactosaminidase had no effect. The polysaccharide stain of isolated proteins with p-phenylenediamine showed a positive reaction. Radiolabeling with [3H]glucosamine did not reveal the 18 kDa and 32 kDa proteins in autoradiography but [3H]galactose did.
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PMID:The characterization of lectin-binding proteins of Chlamydia trachomatis as glycoproteins. 172 47

Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells. The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O-Glycosidically linked chains, containing 70% of the mannose, were released by mild beta-elimination. N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was beta-glycosidically linked. The results indicate that beta-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.
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PMID:Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9. 177 62

Scatter factor (SF), a glycoprotein produced by cultured fibroblasts, acts in vitro on epithelial cells causing separation and increased local motility. In this study, the polypeptide was purified to apparent homogeneity in high yields with conserved biological activity from medium conditioned by ras-transformed NIH 3T3 cells, by a three-step procedure involving ammonium sulphate fractionation, cation-exchange and hydroxyapatite chromatography. After purification, SF specific activity increased from approximately 0.3 units/microgram in unprocessed conditioned medium to approximately 5 units/ng, and cumulative recovery of biological activity was approximately 38%. Treatment of pure SF with N-glycanase resulted in a decreased Mr, but no concomitant effect was observed on biological activity. Proteolytic activity was absent from samples of both partially purified and pure SF. Our biochemical studies showed that SF, which is highly aggregated in low-ionic-strength media, is not aggregated in 0.4 M-salt. Under non-reducing conditions, pure SF migrated as a single stained band at Mr 67,000 on SDS/PAGE, and biological activity was eluted from unstained gels with an identical Mr. SF was electrofocused sharply at pI 8.5 with no degradation of activity. From ultracentrifugation studies (under non-aggregating conditions), the sedimentation coefficient of active SF was 3.7 S and f.p.l.c. molecular sieve chromatography indicated a Stokes' radius of 2.95 nm. The calculated Mr from these data was 61,400. The appearance of three stained polypeptides of Mr 82,000, 57,000 and 32,000 derived from the Mr-67,000 constituent after reduction with mercaptoethanol suggests that SF may be a heterodimer of Mr-57,000 and -32,000 subunits. Data from protein sequence analysis of the hydroxyapatite-purified protein confirms that SF has sequence identity with both rat hepatocyte growth factor and human fibroblast tumour cytotoxic factor.
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PMID:Purification and characterization of biologically active scatter factor from ras-transformed NIH 3T3 conditioned medium. 183 75


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