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)

The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.
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PMID:Identification of the site of sulfation of the fourth component of human complement. 394 9

Experimental conditions required for the expression of maximum C5 activation upon limited trypsin hydrolysis were determined to be 0.008 mol of trypsin/mol C5 in a reaction mixture containing 1 mg C5/ml veronal-buffered saline incubated at 37 degrees C for 30 min. Employing these optimal incubation conditions, the primary or preferred site of trypsin hydrolysis of the C5 alpha-chain resulted in the production of C5 alpha 1 (molecular weight, 90,000) and C5 alpha 5 (molecular weight, 25,000) fragments that remained disulfide bonded to the modified C5 molecule (C5'try). Detailed structural-functional analyses clearly indicated the trypsin-mediated conversion of the C5 alpha-chain to C5 alpha 1 and C5 alpha 5 was responsible for the acquisition of neutrophil lysosomal enzyme-releasing and chemotactic activities. Gel filtration column chromatography under physiological ionic strength, pH 7.4, or in the presence of 0.2% SDS further demonstrated that at least 90% of the total recoverable C5a-like biological activity was mediated by the 210,000 molecular weight forms of trypsin-modified C5. Other physiologically relevant, noncomplement protease enzymes (alpha-thrombin, plasmin, and elastase) also activated C5 to express C5a-like reactivities. Analysis of alpha-thrombin-induced, C5 alpha-chain cleavage events by SDS-polyacrylamide slab gel electrophoresis indicated that the mechanism of alpha-thrombin-activation of C5 is similar to that described for trypsin. Reconciliation of this novel mechanism of C5 activation by trypsin with previously published results, and a discussion of the biological significance of noncomplement enzyme-mediated activation of C5 as it might relate to inflammatory processes in vivo, was presented.
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PMID:Expression of C5a-like biological activities by the fifth component of human complement (C5) upon limited digestion with noncomplement enzymes without release of polypeptide fragments. 622 37

To investigate the greater enzymatic activity of the alternative pathway convertase (and the subsequent greater fixation of C3b) on paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, we have examined the topography of binding of C3b to PNH and normal erythrocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the alpha-chain of C3b was found to bind via predominantly ester bonds to free hydroxyl groups on glycophorin-alpha, the major erythrocyte sialoglycoprotein. The pattern of binding of nascent C3b was the same for normal and PNH erythrocytes. Thus, although C3b binding to a different membrane constituent did not appear to account for the greater enzymatic activity of the alternative pathway convertase when affixed to PNH erythrocytes, it seemed possible that the glycoproteins to which C3b bound might be qualitatively abnormal on the PNH cells, and that structural differences in these molecules might impose modifications in the enzyme-substrate interactions of the alternative pathway convertase. Using methods for radiolabeling both protein and carbohydrate residues, we therefore compared the electrophoretic pattern of the cell-surface glycoproteins on PNH and normal erythrocytes. The glycophorin-alpha dimer was found to be qualitatively abnormal on the PNH cells as evidenced by its greater susceptibility to trypsin-mediated proteolysis. In addition, the abnormal erythrocytes from patients with PNH had fewer periodate oxidizable constituents than did normal erythrocytes, indicating a relative deficiency of cell-surface sialic acid. These investigations suggest that abnormalities in membrane glycoproteins may underlie the aberrant interactions of complement with the hematopoietic elements of PNH.
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PMID:Abnormality of glycophorin-alpha on paroxysmal nocturnal hemoglobinuria erythrocytes. 623 12

Ehrlich ascites cells in mice have been shown to have a cell-surface trypsin-like neutral protease (TLNP) with proteolytic and beta-naphthylamidase activity. This activity is inhibited by low-mol.-wt inhibitors of trypsin but not by 11 high-mol.-wt inhibitors of trypsin in free solution. We believe that lack of inhibition is due to protection given to the enzyme by the chemical environment of the cell surface. These cells were demonstrated to export a collagenase zymogen which has been shown to be activated by the cell-surface TLNP. When this protease was completely inhibited by low-mol.-wt inhibitors of trypsin, chymotrypsin was used to activate the collagenase zymogen exported by Ehrlich ascites cells. Examination of the products of collagenolysis at 15 degrees C demonstrated the expected 3/4- and 1/4-length alpha-chain fragments derived from monomeric collagen, confirming that collagenase was one of the enzymes responsible for lysis of the collagen fibrils in the test system.
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PMID:A trypsin-like neutral protease on Ehrlich ascites cell surfaces: its role in the activation of tumour-cell zymogen of collagenase. 625 67

Tryptase, the dominant neutral protease of human pulmonary mast cell secretory granules, has the capacity in vitro to generate C3a anaphylatoxin from purified human C3. Only the alpha-chain of C3 is cleaved, and major fragments with apparent m.w. of 105,000, 39,500, 34,000, 29,000, and 9000 are detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing conditions. Fragments of 34,000 and 9000 m.w. are detected without reduction. A portion of the 9000 m.w. protein corresponds to C3a by virtue of its co-migration in SDS polyacrylamide gels with purified C3a and with trypsin-generated C3a, by its detection in a radioimmunoassay for C3a, and by its contractile activity on the guinea pig ileum bioassay. In the presence of heparin, another component of the mast cell secretory granule, the rate of appearance and the distribution of C3 cleavage fragments as assessed in SDS polyacrylamide gels are not appreciably changed with the exception that no C3a material can be detected in the SDS polyacrylamide gels or by radioimmunoassay and bioassay of the unresolved reaction mixture. Enhanced catabolism of authentic C3a by tryptase occurs in the presence of heparin and by analogy when C3a is generated from C3 by tryptase in the presence of heparin. Whereas tryptase secreted by activated human mast cells may generate C3a, a potentially important additional mediator of immediate hypersensitivity events, the concomitant release of heparin may serve to down-regulate C3a irrespective of its mechanism of generation.
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PMID:Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase. 633 18

A lamprey protein homologous to the third component of mammalian complement was isolated from lamprey plasma and was tentatively designated lamprey C3. Lamprey C3 is a major protein in lamprey serum with electrophoretic mobility of beta-globulin and with m.w. of 190,000. It consists of three polypeptide chains (84,000-alpha, 74,000-beta, and 32,000-gamma chains) linked by disulfide bonds. The protein retains a unique internal thiolester bond on the alpha-chain that is cleaved on methylamine treatment or on limited proteolysis with trypsin. Lamprey C3 by itself could not bind to zymosan or rabbit red cells, but it could covalently bind to these substances when activated by other factors present in lamprey serum. The binding of lamprey C3 to activating surfaces is mediated by covalent bonds and is accompanied by limited proteolysis of the alpha-chain. A fragment with an m.w. of 35,000 containing the internal thiolester site was isolated from methylamine-treated lamprey C3 bound to activated thiol-Sepharose by extensive tryptic digestion followed by dithiothreitol treatment. Lamprey C3 functions as the essential factor in phagocytosis of rabbit red cells by lamprey phagocytes. However, it is not involved in naturally occurring hemolytic activity in lamprey serum.
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PMID:Purification of a lamprey complement protein homologous to the third component of the mammalian complement system. 649 Dec 86

The procoagulant component has been purified from timber rattlesnake (Crotalus horridus horridus) venom by DEAE-cellulose ion-exchange chromatography followed by affinity chromatography on immobilized p-aminobenzamidine and a final DEAE-Sepharose chromatography. As obtained, the procoagulant gave a single band of Mr 29 500 +/- 2000 on SDS-polyacrylamide gel electrophoresis whether or not the sample was reduced prior to electrophoresis. Schiff's stain indicated the presence of some carbohydrate. The procoagulant showed one predominant and four minor bands on discontinuous gel electrophoresis. All caused fibrinogen solutions to clot. Treatment of the preparation with neuraminidase did not cause the minor bands to comigrate with the major band. Amino acid analysis revealed the presence of eight half-cystines, all of which were present as cystines in the intact molecule. The procoagulant has 13 tryptophans per molecule and an extinction coefficient for a 1% solution at 280 nm of 26.3. This venom procoagulant was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the A alpha-chain of fibrinogen. It was not inhibited by protein trypsin inhibitors, N-ethylmaleimide or dithiothreitol, but it was inactivated by phenylmethylsulfonyl fluoride, indicating an active-center serine. The procoagulant catalyzed the release of negligible acid-soluble peptides from bovine serum albumin, casein and hemoglobin.
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PMID:A thrombin-like enzyme from timber rattlesnake venom. 662 56

By quantitative phosphorus determination on the single chains of human fibrinogen it is demonstrated that the covalently bound phosphorus of adult and fetal fibrinogen is exclusively located in the A alpha chain. The A alpha-chain of fetal fibrinogen contains about twice as much phosphorus as the adult A alpha-chain in the well known position of Ser 3 of fibrino-peptide A as well as in a hitherto unknown second position on the A alpha-chain. By consecutive cleavage of the A alpha-chains of fetal and adult fibrinogen with cyanogen bromide, trypsin, and chymotrypsin, separation of the resulting peptide mixtures and analysis for phosphorylated amino acids, this second phosphorylation site could be traced to Ser 345 of the A alpha-chain. There is only one sequence homology between the two now known in vivo phosphorylation sites of human fibrinogen, namely that the second amino acid to the carboxyl side of the phosphorylated Ser is Glu. The sequence specificity of the up to now unidentified protein kinase phosphorylating fibrinogen allows it to be classified as a member of the group of type-2 casein kinases or casein kinases TS.
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PMID:The location of a second in vivo phosphorylation site in the A alpha-chain of human fibrinogen. 671 96

The rates of proteolytic breakdown for native human hemoglobin (Hb) in CNmet-and oxy-forms, for isolated native alpha- and beta-chains of human Hb with deprotected SH-groups and for apo-Hb--globin at constant temperature 20 degrees as well as for metHb and globin in the temperature range 4-25 degrees were studied. The proteolysis of oxy-forms of proteins was performed in the presence of CN- to prevent the appearance in solution of quickly splitted aqua and hydroxy met-forms. Pepsin (at pH 5.5), trypsin (at pH 7.0 and 8.5) and protease VI (pronase) (at pH 7.0 and 8.5) were used as proteases. The rate of proteolysis was registered simultaneously by proteolysate precipitation in concentrated salt solutions (to determine the content of the native form), by precipitation in aqueous solution of trichloroacetic or perchloric acid and by colouring the terminal NH2-groups by ninhidrin in the total proteolysate. For most cases the data of all the three independent methods fell on a single kinetic curve, each pair protein--protease being represented by their individual curves. Therefore the breakdown of all the protein studied possesses a burst-like ("one-by-one", "all-or-none") character. The protein resistance to the attack by proteolytic enzymes increases in the following order: globin less than oxy-alpha-chain less than metHb less than oxy-beta-chain less than HbO2 congruent to CNmetHb. The use of control repeated proteolysis has made it possible to prove that differences in the rate of proteolytic degradation are not the consequence of spontaneous denaturation of the least unstable forms of proteins in the course of proteolytic reaction but are predetermined by the conformational state of the native macromolecule.
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PMID:[Proteolytic degradation of native hemoglobin and its constituent parts--isolated subunits and globin. I. Kinetic data and the character of the process of the breakdown of native forms]. 681 53

It has been shown previously that methylamine is incorporated into the alpha-chain of human C4, resulting in a loss of haemolytic function and the appearance of a free thiol group in the molecule. In the present study it was demonstrated that a fragment resembling C4d is liberated from C4 by trypsin. The fragment--Try-C4d--contains both the methylamine binding site and the free thiol group. When separated on DEAE-Sepharose, four types of Try-C4d, differing in charge and size, could be defined. The size difference was found to parallel the presence of Chido and Rodgers blood group antigens. Fragments of Mr 30,000 carried the Rodgers antigen and the Chido antigen was expressed on fragments of Mr 28,000.
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PMID:Isolation of tryptic fragments of human C4 expressing Chido and Rodgers antigens. 716 21


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