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

---

Query: EC:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
...
PMID:Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores. 185 49

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.
...
PMID:Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane. 197 26

The RhD polypeptide and LW glycoprotein were separately immunopurified with monoclonal antibodies and compared by two-dimensional (2-D) iodopeptide mapping after digestion with alpha-chymotrypsin. These proteins have distinct 2-D maps, as seen after 125I-labeling tyrosine residues (chloramine-T procedure), and even more strikingly after labeling primary amine residues (Bolton-Hunter procedure). Of the more than 20 iodopeptides visualized, only five migrated identically when preparations of RhD and LW were directly compared, suggesting that RhD and LW are different proteins that may share some common protein domains. N-glycanase treatment of the iodopeptides did not modify the 2-D map of the RhD protein but greatly affected the LW map, further indicating that LW, but not RhD, carries N-linked carbohydrate chains. After deglycosylation the LW map was different from the RhD map, confirming that the RhD and LW polypeptides are different proteins. These findings demonstrate that LW is neither a glycosylated form of Rh protein nor is Rh a precursor of LW.
...
PMID:Comparative analysis by two-dimensional iodopeptide mapping of the RhD protein and LW glycoprotein. 211 34

A rat alpha-amidating enzyme (alpha-AE) cDNA has been expressed in mouse C127 cells using a bovine papilloma virus vector in which transcription was regulated by the mouse metallothionein 1 promoter. The cDNA encoding the full length alpha-AE protein was modified to terminate translation at a site preceding the transmembrane and cytoplasmic domains, thereby enabling functional enzyme to be secreted into the medium. Purification of recombinant alpha-AE to homogeneity indicated that the enzyme was synthesized and secreted as two proteins of 75-77 kDA. The observed heterogeneity was due to inefficient glycosylation at Asn660, as demonstrated by glycopeptidase F digestion. Using the synthetic peptide, dansyl-Tyr-Val-Gly, the specific activity of the recombinant enzyme at pH 7.0 was found to be 1.4 mumol/min/mg and the Km of the enzyme was determined to be 3 microM. The purified recombinant enzyme has maximal activity at pH 4.5-5.5; however, a rapid inactivation of the enzyme occurs in acidic solutions in vitro. This inactivation is diminished when activity is measured at pH 7.0-10.0. The availability of large amounts of readily purified, active recombinant alpha-AE should allow detailed probing of reaction mechanism, copper coordination chemistry, and turn-over-based inactivation events.
...
PMID:Purification and characterization of functional recombinant alpha-amidating enzyme secreted from mammalian cells. 221 57

Freshly isolated rat islets and cultured hamster insulinoma cells (HIT T15) were incubated with a membrane-permeable octanoyl tripeptide (N-octanoyl-ASN-TYR-THR-NH2), which contains an acceptor sequence for ASN-linked glycosylation. Labeled octanoyltripeptide (125[I]TYR) was glycosylated by both islets and HIT cells. The carbohydrate moiety of this glycotripeptide was removed by N-glycanase indicating that glycotripeptide was formed in the lumen of endoplasmic reticulum and, subsequently was secreted via the route for secretory protein. Secretion of glycotripeptide began more rapidly than that of insulin newly synthesized from 3[H]leucine. At 30 min glycotripeptide secretion was already significant but, over a 3-h period, it never represented more than 21% of glycotripeptide produced. Glycotripeptide secretion was not affected by compounds shown to regulate insulin secretion (glucose, forskolin, EGTA and streptozotocin). Thus in beta cells, it appears that glycotripeptide secretion is unregulated and that its cellular secretory pathway is different from that for insulin.
...
PMID:Unregulated secretion of an exogenous glycotripeptide by rat islets and HIT cells. 284 81

Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.
...
PMID:Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W. 289 Mar 46

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.
...
PMID:Biosynthesis of sulfated asparagine-linked complex carbohydrate units of calf thyroglobulin. 338 87

After pepsin digestion, all of the carbohydrates in ovalbumin were recovered in two glycopeptides, Glu-Glu-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val and Glu-Gln-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val. Almond glycopeptidase released quantitatively oligosaccharides from the glycopeptides. The products from both glycopeptides contained both the high-mannose-type oligosaccharides and the hybrid-type oligosaccharides in the same ratio. Thus, either the high-mannose-type or the hybrid-type oligosaccharide is attached to the unique asparagine residue in the ovalbumin molecule.
...
PMID:Either high-mannose-type or hybrid-type oligosaccharide is linked to the same asparagine residue in ovalbumin. 728 36

Structural and functional characteristics of the goat uterine nuclear estrogen receptor R-II have been subjected to comparison with those of the nonactivated estrogen receptor (naER), purified from the cytosol. The two proteins have the same molecular mass, 66 kDa; they display identical peptide maps and are both recognized by anti-estrogen receptor (R-I) IgG. Both are tyrosine kinases and bind with equal affinity to a column of anti-phosphotyrosine IgG-Sepharose. On the other hand, while naER is a glycoprotein, the R-II does not show any sign of glycosylation. Unlike the naER, the R-II is incapable of dimerization with estrogen receptor activation factor (E-RAF) and, as a consequence, bind to the DNA. R-II has a higher estradiol binding capacity and the resultant reduction in its affinity for the hormone in comparison with the naER. Further, the sedimentation behavior and the Stokes radius of the naER indicate a globular nature in the shape of the protein. The corresponding data for the R-II reveal that the protein has a distinct nonglobular shape. Deglycosylation of the naER using a glycopeptidase resulted in the total conversion of the distinct physical features of the naER to the R-II category. This treatment resulted, without effecting any reduction in its molecular mass, in the loss of the E-RAF dimerization capacity of the naER. The Stokes radius and the sedimentation coefficient of the protein underwent drastic changes and became closely similar to those of the R-II. In addition, the deglycosylation introduced a several-fold enhancement in the capacity of the naER to bind estradiol with a concomitant decrease in its affinity, similar to the corresponding properties of the R-II. The R-II is shown to have a conformational structure different from that of the naER, to interact with the nuclear RNA polymerase II. It is also shown here that the R-II phosphorylates two subunits (molecular mass 91 and 20 kDa) in the RNA polymerase II, in addition to the 40-kDa subunit phosphorylated by the naER. The results clearly indicate the possibility that the nuclear R-II estrogen receptor is the deglycosylated naER.
...
PMID:The nuclear estrogen receptor R-II of the goat uterus: distinct possibility that the R-II is the deglycosylated form of the nonactivated estrogen receptor (naER). 754 97

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.
...
PMID:Characterization of the major sulfated protein of mouse pancreatic acinar cells: a high molecular weight peripheral membrane glycoprotein of zymogen granules. 787 32


1 2 Next >>

---