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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A serine protease and a serine protease inhibitor were purified from infective larvae of the parasitic nematode Anisakis simplex. The serine protease was found to be trypsin-like and preferentially cleaved substrates with the basic amino acid arginine at the P1 position (Z-Gly-Pro-Arg-AMC (where Z is benzyloxycarbonyl), Km = 0.019 mM, and Z-Phe-Pro-Arg-AMC, Km = 0.013 mM) at rates similar to those determined for trypsin (0.002 mM and 0.006 mM, respectively). However, the presence of a bulky hydrophobic residue at the P2 position (Z-Phe-Arg-AMC, Km = 13.3 mM, and Z-Ile-Leu-Val-Arg-AMC, Km = 24.7 mM) greatly decreased the rate of substrate hydrolysis. Internal amino acid sequence information was obtained from three endo Lys-C digestion fragments of the purified enzyme. These sequences were > 89% (33:37) identical with that of porcine trypsin. A second serine protease 85% (11:13) identical with that of a secreted tissue-destructive serine protease from the pathogenic bacterium Dichelobacter nodosus was also identified. The serine protease inhibitor was found to inhibit trypsin, elastase, and the Anisakis serine protease stoichiometrically, but did not inhibit chymotrypsin. The amino acid sequence of the amino terminus as well as two internal endo Lys-C fragments were determined. Approximately 96% (47:49) of the residues were identical with soybean trypsin inhibitor, indicating that this inhibitor belongs to the Kunitz-type family of inhibitors.
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PMID:Characterization of the serine protease and serine protease inhibitor from the tissue-penetrating nematode Anisakis simplex. 796 83

DNA sequence analysis of the structural urease genes from Staphylococcus xylosus revealed that three enzyme subunits are encoded in the order of 11,000, 15,400 and 61,000 (mol. mass), which correspond to the single polypeptide chain of jack bean urease (90,800). Comparing the deduced amino acid sequence of S. xylosus urease with the amino acid sequence of jack bean urease an overall portion of 56% identical residues was found. For S. xylosus urease a subunit structure of (alpha beta gamma)4 was proposed, based on the comparison of the deduced amino acid content of the enzyme subunits with the total amino acid content of the purified enzyme. The staphylococcal enzyme contained no cysteine, as deduced from DNA sequence and confirmed by the determination of the total amino acid content in the purified enzyme. Instead of cysteine, known to be catalytically essential in the plant enzyme, and conserved among all bacterial ureases analyzed so far, threonine was found in S. xylosus. This amino acid-exchange was located within a highly conserved domain of 17 amino acids, supposed to be part of the active site. Sequence analysis of the respective region of Staphylococcus saprophyticus urease showed that it also contains threonine instead of cysteine. In contrast to jack bean urease S. xylosus urease was not affected by the SH-group inhibitor dipyridyl disulfide but was completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The presented results indicate that in these staphylococcal strains urea hydrolysis might function in a manner similar to the peptide bond cleavage by chymotrypsin.
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PMID:Threonine is present instead of cysteine at the active site of urease from Staphylococcus xylosus. 804

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.
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PMID:Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor. 811 69

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F. R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF. Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.
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PMID:Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor. 822 33

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.
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PMID:Neutrophil apoptosis induced by proteolytic enzymes. 860 Mar 21

A cysteine-rich serine protease inhibitor (Guamerin II) was isolated from the non-blood sucking leech Whitmania edentula. The new inhibitor was identified as a low molecular weight (6,012 Da) polypeptide with some sequence similarities to antistasin, hirustasin and guamerin. The inhibitor contained 56 amino acid residues with 76.8% sequence similarity to guamerin, 48.2% to hirustasin and 28.6% to the first domain of antistasin. This new inhibitor was the first completely sequenced serine protease inhibitor from a non-blood sucking leech. Analysis of the inhibitor revealed that it was active against neutrophil elastase and chymotrypsin, but had no activity against a variety of other proteases. The P1 reactive site residue was identified as methionine and the residues surrounding the P1 site were hydrophobic amino acids. The primary structure of the inhibitor showed no similarity to well-known elastase inhibitors from leeches such as eglin.
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PMID:A cysteine-rich serine protease inhibitor (Guamerin II) from the non-blood sucking leech Whitmania edentula: biochemical characterization and amino acid sequence analysis. 883 33

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.
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PMID:Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I. 919 29

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.
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PMID:Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator. 964 77

An in vitro normal human epidermal keratinocytes (NHEK) model was used to study and to characterize the protease stimulated by the mustards 2-chloroethyl ethyl sulphide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN2), and Bis-2-chloroethyl sulfide (sulfur mustard, HD). The results obtained by using a chromozym (TRY) peptide substrate protease assay showed the optimum mustard concentration and time for protease stimulation to be about 200 microM CEES, 100 microM HN2 or HD, and 16 hours. The mustard-stimulated protease was membrane-bound, and was inhibited by adding a Ca2+ chelator EGTA (2 mM), BAPTA AM (50 microM) or a serine protease inhibitor diisopropyl fluoro-phosphate DFP (1 mM), or a protein synthesis inhibitor cycloheximide (10 microM) in the extracellular medium. These results suggest that one of the mechanisms of mustard toxicity is via the stimulation of a trypsin/chymotrypsin like serine protease, which is dependent on Ca2+ and new protein synthesis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mustard-stimulated approximately equal to 70-80 KDa protein band that was associated with protease activity which was inhibitable by EGTA, BAPTA, DFP or cycloheximide. This mustard-stimulated protein (protease) may serve as a diagnostic tool for mustard exposure as well as an assay for screening prospective antivesicant protease inhibitor drugs.
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PMID:Protease in normal human epidermal keratinocytes. 970 64


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