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)

A new type of glycopeptidase hydrolyzing beta-aspartylglycosylamine linkages was partially purified from almond emulsin by chromatography on Sephadex G-200 and DE 52. The enzyme degraded stem bromelain glycopeptide, Asn-Asn(Man3,Xyl1,Fuc1,GlcNAc2)-Glu-Ser-Ser, to yield equimolar amounts of intact oligosaccharide, peptide (Asn-Asp-Glu-Ser-Ser), and ammonia. The Km value for the stem bromelain glycopeptide was 4 mM, and the optimum pH was 5.2. The enzyme was markedly inhibited by 10 mM Cu2+, Fe3+, and Zn2+. Thiol inhibitors and actinomycete protease inhibitors had no effect. The glycopeptides used as substrates were prepared from stem bromelain, ovalbumin or ovotransferrin. The enzyme hydrolyzed glycopeptides with 3-11 amino acid residues, whereas it did not hydrolyze glycopeptides with 1-2 amino acid residues. Furthermore, Asn-oligosaccharide was not inhibitory to the enzyme.
...
PMID:Some characteristics of a new glycopeptidase acting on aspartylglycosylamine linkages. 73 97

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.
...
PMID:Tryptase from rat skin: purification and properties. 203 67

Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.
...
PMID:The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase. 242 72

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase. 254 51

Atrial-natriuretic-peptide (ANP) receptor, previously identified as a 140 kDa protein with a disulphide-linked homodimeric structure, was purified from bovine lung by (NH4)2SO4 fractionation and affinity chromatography on ANP-Affi-Gel 10. The purified receptor had a binding capacity of 4.2 nmol of ANP/mg of protein and an affinity constant of 6.5 pM. The isoelectric point of the receptor was 5.8, consistent with the acidic nature of the protein (amino acid analysis revealed a predominance of glutamic acid and aspartic acid residues). Treatment with endoglycosidase H and glycopeptidase F revealed that the receptor has three complex types of oligosaccharide chains per 70 kDa subunit. Deglycosylation of the receptor did not affect its binding activity. Reduction with dithiothreitol and reoxidation by dialysis revealed a strong tendency of the receptor subunits to dimerize via disulphide cross-linking; however, carboxymethylation of the reduced receptor indicated that the intersubunit disulphide bond is not necessary for the ligand-binding activity.
...
PMID:Purification and properties of active atrial-natriuretic-peptide receptor (type C) from bovine lung. 255 6

The beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.
...
PMID:All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase beta-subunit contain oligosaccharide. 283 26

Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.
...
PMID:Active site and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F. 749 89

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).
...
PMID:Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis. 777 20

We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp120) of the HIV-1 BH8 isolate produce by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp120 or of cyanogen bromide-generated fragments of rgp120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential N-glycosylation sites in the rgp120 sequence are utilized. We did not detect N-acetylgalactosamine in rgp120, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by 1H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and N-acetylglucosamine were the only sugars observed in the intact rgp120 and likewise in individual glycopeptides. All glycopeptides derived from rgp120 contained high mannose-type N-linked oligosaccharides, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycosylation sites showed varied degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Man8-9 to GlcNAc2Man5-7. These results demonstrate that N-glycosylation of rgp120 in the baculovirus expression system occurs at all potential sites and is site specific in terms of oligosaccharide structures.
...
PMID:Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system. 821 72

The recent finding of peptide:N-glycanase (PNGase) in medaka embryos (Seko, A., Kitajima, K., Inoue, Y., & Inoue, S., J.Biol. Chem. 266, 22110 (1991)) raised the question of how widespread is the occurrence of this type of de-N-glycosylating enzyme. In experiments designed to identify PNGase in the mammalian system, we searched for its activity in some cultured cell lines. Incubation of a 14C-labeled N-glycopeptide with extracts prepared from cultured cells resulted in producing the free glycan and the peptide. Detailed characterizations of the products, formed upon incubation of a 14C-labeled N-glycopeptide substrate with the enzyme preparation from C3H mouse loose connective tissue-derived L-929 cells, by HPLC, amino acid and carbohydrate composition analyses, and peptide sequence analysis unequivocally established the reaction products to be the free glycan having di-N-acetylchitobiosyl sequence at its reducing end and free peptide in which the originally glycan-linked Asn residue was converted to the Asp residue. This represents the first demonstration of PNGase in mammalian cells and thus PNGase appears to be a very common enzyme expressed in not only plants and bacteria but also a wide range of animals although its functional significance remains to be clarified.
...
PMID:Identification of peptide:N-glycanase activity in mammalian-derived cultured cells. 835 68


1 2 3 4 5 Next >>

---