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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report describes the N-glycosylation site mapping of human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi-alpha(2-->6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn413 and Asn611) in the ratio of approximately 85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl 2,4-triantennary and a trisialyl 2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413 was found to be approximately 5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asn611 in the ratio approximately 1:1.
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PMID:N-glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. 145 41

A gene encoding an alpha-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced. The alpha-1,2-mannosyltransferase which utilizes alpha-methylmannoside as acceptor of mannose from GDP-mannose was purified. The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides. Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA. A specific reaction product was used to screen a genomic library of S.cerevisiae. A fragment of approximately 5.7 kb was isolated, of which a 2.9 kb fragment was sequenced. It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified alpha-1,2-mannosyltransferase. The gene, designated MNT1, is located on the right arm of chromosome 4. It encodes a 442 amino acid polypeptide with a calculated mol. wt of 51.4 kDa. The corresponding mRNA has a length of approximately 1.6 kb. Overexpression of the MNT1 gene increased this alpha-1,2-mannosyltransferase activity approximately 2.5-fold. The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site. The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases. This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.
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PMID:Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene. 155 Sep 92

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.
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PMID:Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies. 161 9

Experiments were carried out to analyze the binding sites on human cells for highly purified retroviral protein p15E isolated from Feline Leukemia Virus, Rickard Strain. Binding of 125I-labeled p15E was tested with surfaces of human peripheral blood lymphocytes and 3 cell lines, Raji, MOLT-4, and U-937. 125I-labeled p15E showed specific binding to human peripheral blood lymphocytes. In addition, all of the cell lines tested showed binding of 125I-labeled p15E. Using U-937 cells, we characterized the interaction between p15E and the surface of these cells, and showed that the binding was specific by the following 3 different sets of evidence: (i) in equilibrium binding experiments, 18,000 binding sites with a dissociation constant of 2 x 10(-9) M were present on U-937 cells; (ii) trypsin or N-glycanase treatment decreased the binding sites of 125I-labeled p15E; and (iii) by affinity chromatography using p15E or BSA Sepharose columns, the isolated membranes of 125I-labeled U-937 cells previously treated with Triton X-100 showed a significantly higher binding to the p15E column than to the BSA column.
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PMID:Specific association of retroviral envelope protein, p15E, with human cell surfaces. 164 29

Female BALB/c mice were immunized with a whole-hyphal-cell extract obtained from Candida albicans wild-type strain 4918 grown in Lee medium. Monoclonal antibody (MAb)-producing hybridomas were prepared by fusing immune splenocytes with NS-1 myeloma cells. One of the hybrid cell clones (1.183) secreted an immunoglobulin G1 antibody that reacted with C. albicans hyphae in an indirect immunofluorescence assay but not with yeast cells and pseudohyphal segments directly originating from parent blastoconidia. In the same assay eight of nine recent clinical C. albicans isolates and Candida stellatoidea tested positive for hyphal cell-specific reactivity with MAb 1.183. The recognized antigen on hyphal cells was sensitive to heat treatment, beta-mercaptoethanol reduction, and proteolysis with pronase, trypsin, and subtilisin. Western blot (immunoblot) analysis of hyphal whole-cell and dithiothreitol extracts with MAb 1.183 revealed two major proteins with approximate molecular masses of 55 and 60 kilodaltons (kDa) under reducing conditions. Endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment reduced the molecular mass of the 60-kDa protein slightly but did not affect recognition by MAb 1.183, whereas peptide:N-glycosidase F (N-glycanase) had no effect on either protein. When exponentially growing yeast cells were treated sequentially with EDTA, beta-mercaptoethanol, and Zymolase to form protoplasts, a specific immunofluorescence signal was obtained with MAb 1.183. In a Western blot, MAb 1.183 showed reactivity with a 20-kDa protein in the sodium dodecyl sulfate extract from protoplasts, whereas no reactivity was found with cell wall material obtained from yeast cells. In summary, these experiments indicated that specific cell surface components from C. albicans hyphae are related to antigens which are present in yeast cells but are not detectable on the surface of the latter.
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PMID:A monoclonal antibody that defines a surface antigen on Candida albicans hyphae cross-reacts with yeast cell protoplasts. 168 99

A hybridoma (3B2-F7) has been established which secretes a monoclonal antibody (Mab) directed against a peptide determinant of human seminal plasma glycoprotein (HSP-gP). The deglycosylation of HSP-gP was performed chemically with TFMS hydrolysis and enzymatically in the presence of detergent and further treated with periodic acid after fixing deglycosylated HSP on plastic wells. The Mab 3B2-F7 (IgM, kappa) exhibited sperm immobilization activity (256 units of SI50) and inhibited sperm binding to human zona pellucida. Human epididymis, pancreatic islets of Langerhan's and distal tubulus of kidney were strongly labelled whilst other tissues were essentially negative by avidin-biotin complex tissue staining with this Mab. The antigen epitope to the Mab was in the 36 kDa molecule of human HSP-gP. The antigenic determinant recognized by Mab 3B2-F7 was destroyed by six different proteases, but was resistant to N-glycanase and other carbohydrate splitting enzymes. This epitope is therefore likely to be composed of a polypeptide chain. Peptide fragments after proteolysis of the HSP molecule with Staph. aureus V8 protease and trypsin retained antigenicity, hence the epitope corresponding to the Mab may be a peptide chain and not dependent on the conformational structure of the polypeptide.
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PMID:Monoclonal antibody recognizing an apparent peptide epitope of human seminal plasma glycoprotein and exhibiting sperm immobilizing activity. 169 87

A Mr 95,000 matrix metalloproteinase (MMP) produced by rat mammary carcinoma cells has been isolated and characterized. The MMP was secreted in a proteolytically inactive form that was free from bound tissue inhibitor of metalloproteinases. The enzyme was highly glycosylated as evident from an apparent drop of Mr from 95,000 to 83,000 after treatment with N-glycanase. Rotary shadowing electron micrographs of purified proenzyme preparations revealed a uniform set of ellipsoidal molecules. Treatment of the proenzyme with 1% SDS resulted in generation of catalytic activity and exposed a cryptic unpaired Cys residue. The latent proenzyme may be activated in at least three additional ways: either spontaneously upon storage, by treatment with organomercurials, or by limited proteolysis by trypsin. Each mode of activation yielded a distinct pattern of cleavage of the enzyme. The activated enzyme cleaved gelatin (denatured type I collagen) and native type IV and V collagen at 30-37 degrees C. Noncollagenous proteins including alpha 1-proteinase inhibitor, casein, and fibrinogen also were cleaved. The rat mammary carcinoma cell line that produces the Mr 95,000 MMP is composed of two distinct (epithelial- and myoepithelial-like) cell types. The enzyme is expressed constitutively by the epithelial cells. This suggests that expression of the Mr 95,000 MMP is regulated differently from that of interstitial collagenase, which is produced by the epithelial cells only in response to specific inductive factor(s) from the myoepithelial-like cells. Monoclonal antibodies raised against the purified latent Mr 95,000 form of the enzyme bind specifically to the Mr 95,000 MMP and have been used to localize the enzyme to the Golgi region and cytoplasmic granules of the epithelial cells.
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PMID:Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells. 199 64

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.
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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.
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PMID:The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase. 242 72

Although antigen-reactive T lymphocytes play a central role in the host response to Histoplasma capsulatum, little is known of the nature of Histoplasma antigens recognized by these cells in vitro. Employing a murine T-cell line and two clones that are reactive with histoplasmin, we examined whether activation of T cells by histoplasmin required the presence of carbohydrate or protein moieties. The approach taken was to modify carbohydrate or protein molecules in histoplasmin by chemical or enzymatic digestion or by lectin adsorption. In parallel, antigen was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate alterations in functional activity with changes in the electrophoretic appearance of histoplasmin. Treatment of histoplasmin with periodate (0.1 M, 0.05 M, and 0.01 M) or with the endoglycosidases N-glycanase and endoglycosidase H sharply diminished the capacity of histoplasmin to trigger responses by T cells. Reactivity of T cells to histoplasmin that had been adsorbed with lectins binding mannose, glucose, or galactose was reduced by greater than 70%; conversely, the responses by T cells to antigen that had been adsorbed with lectins specific for fucose, N-acetylgalactosamine, or N-acetylglucosamine ranged from 82 to 91% of that to control antigen. Proliferative responses by T cells to histoplasmin that had been digested with chymotrypsin, protease, or trypsin were 2 to 43% of control values. The electrophoretic appearance of histoplasmin was modified by some but not all of the treatments. Partially purified H and M antigens triggered proliferation of T cells. Thus, both carbohydrates and proteins must be present to induce optimal responses by T cells. A portion of the carbohydrates is N linked to proteins, and alpha-D-mannose (or alpha-D-glucose) and beta-D-galactose are the sugar ligands of carbohydrate-containing antigens.
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PMID:Characterization of antigenic determinants in histoplasmin that stimulate Histoplasma capsulatum-reactive T cells in vitro. 245 54


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