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)

Using affinity chromatography with the monoclonal antibody 271A6, which binds selectively to telencephalic regions of the rabbit brain, we have purified a telencephalon-specific antigen to apparent homogeneity and characterized it as a membrane glycoprotein. The telencephalon-specific membrane protein (named "telencephalin") has a molecular weight of about 500,000 and is composed of four subunits each of mol. wt 130,000. Its digestion with N-glycanase reduced the subunit mol. wt by 23,000, indicating that each subunit has several N-asparagine-linked oligosaccharide chains. Immunohistochemical analysis using polyclonal antibody against the purified telencephalin shows that expression of the entire protein is restricted to the telencephalon. In addition, segment-specific expression of telencephalin was observed in all mammalian species examined (mouse, rat, guinea-pig, rabbit, cat and monkey). The telencephalon is the most rostral segment of the brain, and comprises the cerebral neocortex, paleocortex, hippocampus, septum, striatum and olfactory bulb. The present results indicate that all regions of the mammalian telencephalon express the segment-specific membrane glycoprotein, telencephalin, and suggest that telecephalin is involved in functions specific to the surface membrane of telencephalic neurons.
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PMID:Mammalian telencephalic neurons express a segment-specific membrane glycoprotein, telencephalin. 235 99

The neuronal membrane protein which binds the K+-channel ligands dendrotoxin, mast cell degranulating peptide, and beta-bungarotoxin was purified from rat brain membranes. When analysed on 10% SDS gel electrophoresis, the purified protein contained two peptides: the toxin-binding subunit of apparent Mr 90,000 and another peptide of Mr 38,000. Neuraminidase treatment reduced the Mr of the toxin-binding subunit to 70,000. Glycopeptidase F gave a further reduction to Mr 65,000. In contrast, the peptide of Mr 38,000 showed no change in Mr upon treatment with neuraminidase and/or glycopeptidase F. It is concluded that the toxin-binding subunit of the dendrotoxin-binding protein, a presumptive K+ channel, is a sialated membrane protein with a peptide core of, at most, Mr 65,000.
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PMID:Enzymatic deglycosylation of the dendrotoxin-binding protein. 270 49

NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.
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PMID:Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity. 278 34

We previously reported that the immunohistochemically defined LFA-1 antigen (LFA-1-like antigen) was expressed on various exocrine tissues uninvolved with tumors in patients with malignant diseases using LFA-1 alpha-specific monoclonal antibodies (mAb) 2F12 and HVS6B6. In this study we investigated differences at the molecular level between LFA-1 on leukocytes and LFA-1-like antigen on MKN45.16, a subline derived from an adenocarcinoma line MKN45 that expresses a high amount of LFA-1-like antigen. LFA-1-like antigen was reactive to mAb 2F12 or HVS6B6, but not to any of the other five different LFA-1 alpha (CD11a)-specific or four LFA-1 beta (CD18)-specific mAb. mAb 2F12 immunoprecipitated a 200-kDa membrane protein (LFA-1-like antigen) from MKN45.16 cells, whereas it precipitated 180-kDa (LFA-1 alpha) and 95-kDa (LFA-1 beta) proteins from a monocytic cell line (THP-1) under both nonreducing and reducing conditions. The molecular difference was confirmed further by N-glycanase treatment of the immunoprecipitates. The isoelectric point of LFA-1-like antigen was 6.0, whereas those of LFA-1 alpha and LFA-1 beta were 6.0 and 4.7, respectively, by two-dimensional gel electrophoresis. Expression of LFA-1 alpha gene on MKN45.16 cells was not detected at the mRNA level by six different sets of LFA-1 alpha-specific oligonucleotides and reverse transcription-polymerase chain reaction. These results indicated that LFA-1-like antigen on an adenocarcinoma cell line was a distinct molecule from LFA-1 on leukocytes.
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PMID:Immunohistochemically defined lymphocyte function-associated antigen 1 (LFA-1) on an adenocarcinoma cell line is a distinct molecule from LFA-1 on leukocytes. 749 27

Ammodytoxin A, the presynaptic neurotoxin from Vipera ammodytes ammodytes venom, was found to bind specifically and with high affinity to bovine cortex synaptic membrane preparation. The detected ammodytoxin A high-affinity binding was characterized by equilibrium binding analysis which revealed a single high-affinity binding site with Kd 4.13 nM and Bmax 6.67 pmoles/mg of membrane protein. 125I-ammodytoxin A was covalently cross-linked to its neuronal acceptor using a chemical cross-linking technique. As revealed by subsequent SDS-PAGE analysis and autoradiography, 125I-ammodytoxin A specifically attached to membrane components with apparent mol. wts 53,000-56,000. Besides by the native ammodytoxin A, the binding of radioiodinated ammodytoxin A to the neuronal acceptor was highly attenuated, also by other two iso-neurotoxins from V. a. ammodytes venom, ammodytoxins B and C, and neurotoxin crotoxin B from the venom of the South American rattlesnake (Crotalus durissus terrificus). Vipera berus berus phospholipase A2 was a weaker inhibitor, whereas nontoxic phospholipase A2, ammodytoxin I2 and myotoxic phospholipase A2 homologue, ammodytin L, both from V. a. ammodytes venom as well, were very weak inhibitors. No inhibitory effect on 125I-ammodytoxin A specific binding at all was, however, obtained with alpha-dendrotoxin, beta-bungarotoxin and crotoxin A, respectively. Treatment of synaptic membranes with proteinase K and Staphylococcus aureus V-8 proteinase, a combination of PNGase F and neuroaminidase, heat or acid lowered the 125I-ammodytoxin A specific binding to various extents but never completely abolished it. The ammodytoxin A binding site in bovine synaptic membranes is thus most likely a combination of membrane glycoprotein acceptor and membrane phospholipids. As ammodytoxin A reduced the second negative component of the perineural waveform, measured on mouse triangularis sterni preparation, which is very likely a result of an inhibition of a fraction of the terminal K+ currents, the ammodytoxin A acceptor could well be connected with K+ channels.
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PMID:Ammodytoxin A acceptor in bovine brain synaptic membranes. 757 Jun 29

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 Epstein-Barr virus (EBV) major membrane protein, gp350, induces antibodies that neutralize virus infectivity in vitro and is a potential candidate for an EBV vaccine. Full-length EBV gp350 and five protein fragments, encompassing the entire protein sequence, were generated in a baculovirus expression system. The recombinant proteins were analysed using a panel of 14 monoclonal antibodies (MAbs) (13 prepared against native gp350 derived from virus-producing cells and one prepared against an Escherichia coli recombinant protein). All 14 MAbs, including a virus-neutralizing antibody, reacted with the full-length recombinant gp350 in a dot blot immunoassay, but only four of the 14 MAbs reacted with polypeptides expressed by the five subclones, indicating that the full-length protein, but not the protein fragments, was antigenically similar to native gp350. Treatment of the six recombinant proteins with peptide-N-glycosidase F (PNGase F) indicated that the full-length gp350 protein and the N-terminal fragment were glycosylated and that the four internally initiated polypeptides were not glycosylated. PNGase F treatment of the full-length glycosylated gp350 did not eliminate its reactivity with all of the 10 MAbs examined (including the neutralizing MAb) in a dot blot immunoassay; however, denatured glycosylated gp350 lost reactivity with all but four of the 14 MAbs when analysed by either dot blot or Western blot immunoassay. The data suggest that conformational epitopes are more important in recognition of gp350 by this panel of MAbs than glycosylation sites, and that the epitope on gp350 recognized by the neutralizing MAb is conformation- and not glycosylation-dependent.
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PMID:Conformation-dependent recognition of baculovirus-expressed Epstein-Barr virus gp350 by a panel of monoclonal antibodies. 769 88

The major sulfated protein of the mouse pancreatic acinar cell, gp300, has been identified and characterized with monoclonal and polyclonal antibodies. gp300 is a glycoprotein of M(r) = 300,000 which contains approximately 40% of metabolically incorporated [35S]sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis, demonstrating that the sulfate is carbohydrate-linked rather than tyrosine-linked. gp300 metabolically labeled with [3H]glucosamine and [35S]sulfate was chemically and enzymatically treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycans. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released approximately 17% of [3H] and little [35S]. Mild alkaline borohydride treatment after removal of N-linked sugar released the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthesis studies and PNGase F digestion indicate that the core protein is approximately 210 kDa, with apparent contributions of approximately 35 kDa N-linked sugar, and approximately 55 kDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presence of Gal beta(1-3)GalNAc and sialic acid alpha(2-3)Gal in O-linked oligosaccharide, and Gal beta(1-4)GlcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral membrane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in response to hormone stimulation of acini, so it is not a secretory product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule biogenesis.
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PMID:Characterization of the major sulfated protein of mouse pancreatic acinar cells: a high molecular weight peripheral membrane glycoprotein of zymogen granules. 787 32

The 32-kDa glycoprotein of Chlamydia trachomatis was shown to have a pI of 6.2 to 6.4 which distinguished this protein from the chlamydial histone-like protein of similar molecular mass that has a pI of > 10. The initial interaction of the glycan of 32 kDa glycoprotein and HeLa cells was also investigated. Glycan was cleaved from the protein backbone by N-glycanase and radiolabeled with tritium by sodium borohydride reduction. Competition assays showed the binding of glycan to HeLa cells was inhibited by galactose, mannose, and N-acetylglucosamine but not by sedoheptulose and fructose. Untreated and UV-treated organisms inhibited the binding, while heat-inactivated organisms did not. Binding was blocked by rabbit antiserum against whole organisms but not by rabbit anti-155-kDa antiserum or monoclonal antibodies against the lipopolysaccharide and major outer membrane protein.
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PMID:The 32-kDa glycoprotein of Chlamydia trachomatis is an acidic protein that may be involved in the attachment process. 798 77

P-glycoprotein (P-gp) is a highly-conserved membrane protein expressed in various multidrug-resistant cell lines. P-glycoprotein was detected in capillaries isolated from human, beef and rat brains with a Western immunoblotting procedure using the monoclonal antibody C219 (mAb C219) specific for P-gp. The mAb C219 detected a 180 kDa protein in brain capillaries isolated from all three species. The largest amount of antigen was detected in capillaries isolated from human brain. Specific binding was assessed by competitive inhibition of mAb C219 binding by the synthetic epitope VQEALD. The glycoprotein nature of the brain capillary proteins was confirmed by its sensitivity to N-glycanase treatment, which reduced their apparent molecular mass by 5 to 10 kDa. In addition, immunohistochemical studies using the antibodies C219, JSB-1 and C494 were performed. Bovine and rat capillaries showed reactivity only with the mAb C219. Heavy staining of human brain capillaries was observed with both antibodies C219 and JSB-1, while only weak staining was observed with antibody C494. These results clearly show that P-glycoprotein is strongly expressed at the blood-brain barrier (BBB) site and suggest that this protein may play a physiological role in regulating the access of certain molecules to the central nervous system, or in the secretory functions of the BBB.
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PMID:High levels of P-glycoprotein detected in isolated brain capillaries. 810 51


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