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)

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

Kidney microsomes were fractionated with Triton X-114, to give a fraction enriched in the renal tubule H(+)-translocating ATPase, as judged by the sensitivity of its ATPase activity to bafilomycin A1, and its content of two polypeptides recognized by antibodies directed against subunits of plant tonoplast ATPases. This fraction contained a polypeptide of apparent molecular mass of 115 kDa, that was recognized by an antibody to the largest (120 kDa) subunit of chromaffin-granule membrane H(+)-ATPase, and, like this subunit, was reduced in molecular weight on treatment with glycopeptidase F. We conclude that, like other mammalian vacuolar H(+)-ATPases, the kidney H(+)-ATPase contains a large, glycosylated subunit.
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PMID:The vacuolar H(+)-translocating ATPase of renal tubules contains a 115-kDa glycosylated subunit. 182 13

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

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.
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PMID:Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores. 185 49

Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.
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PMID:Localization and structure of the asparagine-linked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule. 185 26

Fucosidosis is an inherited lysosomal storage disease due to a deficiency of alpha-L-fucosidase activity. Exponentially growing lymphoid cell cultures from a fucosidosis patient (JH) had 16-fold lower extracellular alpha-L-fucosidase protein and 72-fold lower intracellular alpha-L-fucosidase protein with negligible catalytic activity as compared with the mean of 19 control cultures. The percentage of total alpha-L-fucosidase protein released extracellularly by JH cells was 71% as compared with 35% +/- 9% for control cells. During a 1.5 h pulse with 35S-methionine, alpha-L-fucosidase was synthesized by JH cells as an intracellular doublet with Mr of 58,000 and 56,000 and by control cells as an intracellular form with Mr = 58,000. During a subsequent 21 h chase with unlabeled methionine, JH alpha-L-fucosidase was entirely secreted. In contrast, only 25%-30% of control enzyme was secreted with the remainder retained intracellularly. Thus, JH lymphoid cells synthesized a reduced amount of alpha-L-fucosidase that was catalytically inefficient and was hypersecreted. Treatment of JH alpha-L-fucosidase with N-glycanase produced polypeptide chains with Mr of 52,000 and 54,000. Previously, treatment of control alpha-L-fucosidase with N-glycancase produced a single polypeptide chain with Mr of 52,000 (Biochem Genet 1988; 26: 401-20). The doublet polypeptide chains of alpha-L-fucosidase in JH cultures may represent expression of two distinct allelic forms of mutant alpha-L-fucosidase.
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PMID:Defective expression of alpha-L-fucosidase by lymphoid cells of a fucosidosis patient. 187 10

The carbohydrate composition and the immunoreactivity of the S and M glycoproteins of the coronavirus TGEV were studied at different stages of their maturation. The biosynthesis of S and M was analyzed in the presence of tunicamycin and monensin. The effect of treatment with endoglycosidases H and F and glycopeptidase F on the precursors and mature forms of S and M were also examined. Species 175K and 29K were characterized as high mannose forms of S and M, respectively, and species 220K and 30-36K as complex type glycosylated forms of these two proteins. M was present mainly as a 29K species in mature virions whereas the 175K form of S was not detected, thus implying that the two proteins undergo Golgi modifications at a far different efficiency. Anti-S and -M monoclonal antibodies were examined for their reactivity towards polypeptide species either treated with endo H or produced in the presence of tunicamycin. It was found that (i) among the four major antigenic sites previously defined (Delmas et al., 1986), only site C (amino acids 363 to 371) was notably expressed by the unglycosylated S polypeptide 155K, whereas the three other sites were dependent upon core-glycosylation, (ii) three of the four anti-M mAbs tested did not recognize the unglycosylated M polypeptide 26K. These data led us to conclude that co-translational, but not terminal glycosylation is an essential requirement for both acquisition and maintenance of the antigenicity of TGEV glycoproteins.
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PMID:Carbohydrate-induced conformational changes strongly modulate the antigenicity of coronavirus TGEV glycoproteins S and M. 195 Jan 69

We isolated 7.4 mg of pure renin from 2 kg of rat kidneys using affinity chromatography on pepstatin-aminohexyl-Sepharose and an octapeptide renin inhibitor, H-77-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that renin consists of two polypeptide chains linked by a disulfide bond, one of Mr = 36,000 (heavy chain) and the other of Mr = 3,000 (light chain). The amino-terminal 10-amino acid sequences of the heavy and the light chains were identical to the sequences beginning at Ser72 and Asp355, respectively, of the amino acid sequence of preprorenin deduced from the renin cDNA sequence. Amino acid sequencing of the carboxyl-terminal peptide of the heavy chain, generated by digestion with lysyl endopeptidase, showed that the carboxyl-terminal residue of the heavy chain is Phe. Thus, the propeptide of prorenin is cleaved after Thr71, followed by removal of two amino acids, Arg353 and Asn354, the result being formation of the heavy and light chains. Thus, the site of cleavage of rat prorenin is after a nonbasic amino acid, in contrast to the cleavage of the propeptide after a pair of basic amino acids in mouse submaxillary renin, human renal renin, and many secretory proteins. Treatment of renin with neuraminidase or glycopeptidase F had no apparent effect on the charge heterogeneity of renin. Glycosylation probably does not contribute to charge heterogeneity.
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PMID:Amino-terminal amino acid sequence and heterogeneity in glycosylation of rat renal renin. 201 14

We previously described the production of monoclonal antibodies against a preparation of membrane glycoproteins from human brain [Berglund et al. (1987) J. Neurochem. 48, 809-815]. One of the glycoproteins, recognized by monoclonal antibody CF3, was specifically expressed in the brain. We now report the isolation and characterization of this glycoprotein, called glycoprotein 135 (Gp135). Gp135 was purified by means of lentil lectin affinity chromatography and immunoaffinity chromatography, using monoclonal antibody CF3, from a crude membrane extract of human brain cortex. Gp135 was shown to consist of a glycosylated single polypeptide chain with an apparent molecular mass of 135 kDa. The size of the polypeptide moiety was estimated to 115 kDa following N-glycanase digestion. The glycoprotein is anchored in the membrane by a glycosylphosphatidylinositol tail, as shown by phospholipase C digestion and liposome incorporation experiments. Amino acid sequence analysis of the amino terminal, and of an internal peptide obtained by V8 protease digestion of the glycoprotein, revealed a strong similarity to three previously described glycoproteins from chicken (contactin and F11) and mouse (F3) brains. These glycoproteins belong to the immunoglobulin superfamily and are implicated in cell adhesion phenomena in the developing brain. Gp135 may be the human counterpart to one or several of these glycoproteins.
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PMID:Isolation and characterization of a membrane glycoprotein from human brain with sequence similarities to cell adhesion proteins from chicken and mouse. 202 73

Basigin is a new member of the immunoglobulin superfamily with homology to both the immunoglobulin V domain and major histocompatibility complex class II antigen beta-chain. Southern blot analysis indicated that the basigin gene was present as a single copy or as a few copies per mouse genome. Although a homologous gene was detected in the hamster and human, Southern and Northern blotting experiments indicated considerable species specificity in the basigin structure. The molecular weight of N-glycanase-treated basigin from embryonal carcinoma cells was about 32,000 and was close to the value of basigin polypeptide inferred from the cDNA sequence; the result confirmed the open reading frame of basigin. Upon Western blotting, large amounts of basigin were detected in the mouse kidney as a glycoprotein bound to Ricinus communis agglutinin (RCA)-I and as a glycoprotein bound to concanavalin A; the molecular weight of the former was 38,000-43,000, and of the latter was 30,000. Basigin of the molecular weight of 48,000 was detected in RCA-I-binding glycoproteins of the liver, small intestine and spleen. Thus, different forms of basigin can be produced by different modes of glycosylation. Another source of heterogeneity of basigin may be differences in N-terminal sequences, since cDNA clones with different 5' coding sequences were identified.
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PMID:Basigin, a new member of the immunoglobulin superfamily: genes in different mammalian species, glycosylation changes in the molecule from adult organs and possible variation in the N-terminal sequences. 203 6


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