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)

Subgroup B adenoviruses (Ad3, -7, -11, -35) contain two open reading frames (ORFs) in the early E3 transcription unit that are not present in subgroup C adenoviruses (Ad2, Ad5). The product of one of these ORFs, a 20,500-kDa (20.5K) protein, was shown previously to be expressed as two diffuse 22K and 36K bands on SDS-PAGE; the 22K appeared to be the precursor to the 36K species. As judged by its predicted sequence, 20.5K is a type I membrane glycoprotein with two potential sites for N-glycosylation and a transmembrane domain near its COOH-terminus. Here we show that when Ad3- or Ad7-infected cells were radiolabeled in the presence of tunicamycin, which prevents the addition of N-linked oligosaccharides, both the 22K and the 36K forms of 20.5K showed increased mobility in SDS-PAGE, indicating that both forms contain N-linked sugars. Both the 22K and the 36K forms were sensitive to digestion by endoglycosidase F and N-glycanase, again indicating that they both contain N-linked sugars. Only the 22K species was sensitive to endoglycosidase H, indicating that it contains high-mannose-type oligosaccharides and that the 36K species contains complex-type carbohydrates. The 36K form was sensitive to neuraminidase, indicating that its sugars contain terminal sialic acid. When digested with N-glycanase and neuraminidase, the 36K form was sensitive to O-glycanase, indicating that the 36K form has O-linked oligosaccharides. The 22K form was labeled with [3H]mannose and the 36K form was labeled with [3H]glucosamine and to a much lesser extent by [3H]mannose. Altogether these results indicate that the 20.5K protein is cotranslationally modified with N-linked high-mannose oligosaccharides, then the protein moves into the Golgi and trans-Golgi network where it acquires O-linked and complex N-linked oligosaccharides.
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PMID:The E3-20.5K membrane protein of subgroup B human adenoviruses contains O-linked and complex N-linked oligosaccharides. 761 71

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.
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PMID:Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells. 764 Feb 9

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

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavenis (himehabu snake) venom, released specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for alpha-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI = 8.1) than okinaxobin I (pI = 5.4). The N-terminal sequence was highly similar to those of okinaxobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like alpha-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to alpha-thrombin.
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PMID:Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurus okinavensis (himehabu snake) venom which release fibrinopeptides A and B. 772 19

We isolated erythropoietin (Epo) from anemic-rat serum with 1.3 x 10(6)-fold purification and 38% recovery using immunoaffinity chromatography. The isolated Epo migrated in SDS polyacrylamide gel with a molecular size of 37 kDa. Biological properties of rat Epo were compared with those of human Epo using target cells of primate and murine origins. When murine cells were used as target cells for assaying Epo, rat Epo stimulated proliferation of the cells with a 50% lower potency than did human Epo. The activity of rat Epo on human cells was only 25% of that of human Epo. Studies of Epo binding to the receptor indicated that rat and human Epos were not distinguishable in binding to murine cells; however, rat Epo bound to the receptor on human cells with an affinity much lower than that of human Epo. Rat Epo was digested with N-glycanase. Complete removal of N-linked sugars converted the native Epo to the deglycosylated form with 18 kDa. The in vitro activity of deglycosylated Epo was 2.5-fold higher than that of the native Epo.
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PMID:Characterization of erythropoietin isolated from rat serum: biochemical comparison of rat and human erythropoietins. 776 37

Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).
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PMID:Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis. 777 20

The isolation and partial characterization of PAS-4 glycoprotein (78 kDa) from bovine milk fat globule membrane (MFGM) is described. PAS-4 was selectively extracted with Triton X-114 nonionic detergent and then fractionated on DEAE-Sepharose at pH 7.5. The PAS-4 fraction that was not bound on DEAE-Sepharose gave a single band by SDS-PAGE. The recovery of PAS-4 was 57.4% from MFGM. An amino acid analysis found a high percentage of nonpolar residues. Approximately 7.2% of carbohydrate from PAS-4 was composed of mannose, galactose (Gal), N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and sialic acid, most of the Gal and GalNAc in PAS-4 being released after mild alkaline hydrolysis. This indicated that PAS-4 contained both N- and O-linked sugar chains in concordance with the results of lectin affinity. PAS-4 had apparent isoelectric points of 7.45, 7.41, and 7.32, but these were shifted to pI 7.47 by a neuraminidase treatment. The apparent molecular weight of PAS-4 after deglycosylation with N-glycanase was approximately 57,000 by SDS-PAGE.
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PMID:Rapid and simple procedure for purifying PAS-4 glycoprotein from bovine milk fat globule membrane. 778 99

Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.
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PMID:Purification and characterization of two forms of beta-D-galactosidase from rat epididymal luminal fluid: evidence for their role in the modification of sperm plasma membrane glycoprotein(s). 782 52

Heparan sulfate 6-sulfotransferase, which catalyzes the transfer of sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified 10,700-fold to apparent homogeneity with a 40% yield from the serum-free culture medium of Chinese hamster ovary cells. The isolation procedure included affinity chromatography of the first heparin-Sepharose CL-6B column (stepwise elution), 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradient elution). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 52 and 45 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the amino group of glucosamine residue or to position 2 of the iduronic acid residue. Heparan sulfate was also sulfated by the purified enzyme at position 6 of N-sulfoglucosamine residue. Chondroitin and chondroitin sulfate did not serve as acceptors. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.44 microM.
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PMID:Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells. 787 70

Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.
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PMID:Analysis of carbohydrates on IgG preparations. 789 Dec 93


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