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)

The inhibitory mechanism of trans-4-aminomethylcyclohexanecarbonyl-L-phenyl-alanine-4-carbo xymethylanilide (1), a noncovalent serine protease inhibitor synthesized based on previous structure-activity studies, was clarified based on the X-ray crystal structure of the complex (2.2 A resolution, R = 0.175), where the amino group of the aminomethylcyclohexane moiety was bifurcately hydrogen-bonded to the carboxyl oxygens of Asp 189 side group (specificity pocket), and the hydrogen bonds of the cyclohexanecarbonyl oxygen to NHs of Gly 193 and Ser 195 residues (oxyanion hole) and of Phe NH to Ser 195 O gamma atom (catalytic triad) were observed. In contrast, the Phe benzene moiety and terminal carboxymethylanilide of 1 were not well located on the electron density map, suggesting the conformational freedom of these P1' and P2' sites at the binding pocket. Based on these insights, trans-4-aminomethylcyclohexanecarbonyl-4-nitro-L-phenylalanine-4-+ ++benzoylanilide (2) was designed, in which the P1' and P2' sites were modified so as to effectively interact with the amino acid residues of trypsin binding pocket via hydrogen bonding and van der Waals interactions, respectively. Consequently, 2 showed 40 times higher inhibitory activity against trypsin than 1.
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PMID:Design of noncovalent trypsin inhibitor based on the X-ray crystal structure of the complex. 764 15

Two chondroitin sulfate-containing complexes have been isolated from fetal bovine serum and shown to contain the serine protease inhibitor bikunin. A complex of 126 kDa contains bikunin linked by a chondroitin sulfate chain to a protein with homology to the HC2 component of the human inter-alpha-trypsin inhibitor. This complex represents the extracellular matrix stabilizing factor recently described as a bikunin-containing fraction necessary for expansion of the cumulus matrix [(1992) J. Biol. Chem. 267, 12380-12386]. A second complex of 236 kDa contains, in addition to bikunin and HC2, a bovine homolog of HC3 of the human pre-alpha-trypsin inhibitor. Thus, bovine bikunin is a chondroitin sulfate proteoglycan that achieves multifunctionality by linkage to proteins homologous to human serine antiproteinase complexes.
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PMID:Subunit structure of bovine ESF (extracellular-matrix stabilizing factor(s)). A chondroitin sulfate proteoglycan with homology to human I alpha i (inter-alpha-trypsin inhibitors). 768 11

The replication of LLC-MK2-grown noninfectious Sendai virus, containing exclusively fusion (F) glycoprotein precursors, was examined in the mouse lung to study the accessibility of virus inoculated intranasally to the virus activator present in the lung. When mice were intranasally inoculated with various doses of the virus after in vitro activation with trypsin, the 50% mouse infectious dose (MID50) was determined to be 0.7 cell-infectious units (CIU) per mouse, indicating that one infectious unit of Sendai virus is enough to initiate replication in the mouse lung and that the present experimental system is highly sensitive. On the other hand, in mice inoculated with virus not treated with trypsin, virus replication in the lung was recognized even in mice inoculated with samples containing no infectious virus, and the MID50 was determined to be 67.5 CIU per mouse (here, CIU were assayed after in vitro trypsin treatment). When mice were infected with 20 MID50 of trypsin-treated infectious and untreated noninfectious viruses (an approximately 100-fold greater amount of noninfectious virus than of infectious virus was used), the noninfectious virus was found to require 2 more days of incubation than the infectious virus, and many of the F proteins synthesized in the lungs of mice infected with the F0-containing virus were present in the cleaved form. In addition, the infection of mice with noninfectious virus was strongly suppressed by aprotinin, a serine protease inhibitor. These results indicate that Sendai virus can initiate replication in the mouse lung even with the F0-containing noninfectious virus and strongly suggest that this infection process is mediated by cleavage activation of the F0 proteins of inoculated viruses by a serine protease(s) present in the lumen of the mouse respiratory tract but that activation of the noninfectious virus is an inefficient process.
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PMID:F0-containing noninfectious Sendai virus can initiate replication in mouse lungs but requires a relatively long incubation period. 769 76

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity.
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PMID:Isolation of a high affinity inhibitor of urokinase-type plasminogen activator by phage display of ecotin. 774 76

Contrapsin, a serine protease inhibitor (serpin) present in mouse serum, was compared with that found in adult Schistosoma mansoni worm homogenates, which although immunologically identical to contrapsin in mouse serum, had a higher molecular weight in Western blotting. Immunolocalization studies demonstrated parasite-associated contrapsin on the surface and interstitial cells of adult male worms. After extraction of these parasites with Triton X-114, contrapsin was found in the aqueous phase of the detergent, suggesting it is unlikely to be an integral membrane protein. Treatment of adult worms with deoxycholate resulted in a change in the electrophoretic behaviour of worm-derived contrapsin. Parallel studies with trypsin suggested this was due to interaction of the serpin with a protease. Using porcine pancreatic trypsin as a model for a putative schistosome protease reacting with contrapsin, we have shown that trypsin, following complex formation with contrapsin, loses immunogenicity. Thus, when contrapsin-trypsin complexes were used as immunogen, the resulting antisera contained antibodies to contrapsin and contrapsin-trypsin complexes only, and none to native trypsin. Thus, epitopes characterizing native trypsin were presumably either masked following complex formation with contrapsin, or their processing and presentation to antigen presenting cells was suppressed, so that an antibody response was not mounted against them. These observations encourage speculation that S. mansoni may be elaborating an immune evasion strategy whereby immunologically sensitive proteases are first complexed with host serpins, which would render them immunogenically inert, and then cleared from the circulation by the host's reticulo-endothelial system. In this way the immune system would be unable to 'see' sensitive parasite proteases sufficiently to mount a response against the parasite.
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PMID:Schistosoma mansoni host-parasite relationship: interaction of contrapsin with adult worms. 780 Apr 17

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema. Equivalent substitutions have been described in alpha 1-antitrypsin and antithrombin III. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional. Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor. A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val451-->Met and Pro476-->Ser mutant proteins, and to a lesser extent Phe455-->Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val451-->Met and Pro476-->Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val451-->Met or Pro476-->Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxylterminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.
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PMID:COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization. 785 21

A cDNA clone, pCP9, has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp alpha 1-antitrypsin. This clone is 1,396 bp in length and has an open reading frame encoding a protein of 410 amino acid residues. The deduced amino acid sequence shows moderate homology to human alpha 1-antitrypsin (38%), guinea pig contrapsin (35%), human alpha 1-antichymotrypsin (34%), and human proteinase C inhibitor (31%), all members of the serine protease inhibitor (serpin) family. To confirm further that the cDNA clone was derived from the authentic carp alpha 1-antitrypsin gene, the presumptive mature protein of pCP9 was expressed in Escherichia coli. The molecular mass of the recombinant protein matched that predicted from the nucleotide sequence. This recombinant protein, which was recognized by antiserum against native alpha 1-antitrypsin, was capable of formation of serpin-enzyme complexes with trypsin, chymotrypsin, and elastase. Therefore, we conclude that the protein encoded by the pCP9 clone is indeed carp alpha 1-antitrypsin. Expression of alpha 1-antitrypsin in brain was confirmed by reverse transcription and polymerase chain reaction performed on mRNA derived from both common carp and bighead carp brain.
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PMID:A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: II. cDNA cloning, sequence analysis, and Escherichia coli expression. 789 Nov

Antithrombin III (ATIII) is a member of the serine protease inhibitor (serpin) family. As a step towards a better understanding of the heparin-binding mechanism of mammalian ATIIIs, the amino acid sequence of porcine ATIII was established by sequence analysis of the peptides derived from cyanogen bromide cleavage and enzymatic digestion with lysyl endopeptidase, V8 protease, and trypsin. Porcine ATIII was found to consist of 431 amino acid residues, with a calculated molecular weight of 48,930 without carbohydrate. Its molecular weight with 16.4% carbohydrate was estimated as 56,955, which is in good agreement with the value determined by SDS-PAGE. Porcine ATIII showed high sequence similarity to other mammalian ATIIIs, including the reactive site, heparin-binding basic amino acid residues, and disulfide bonds. The most notable feature of porcine ATIII was that it possesses only three carbohydrate chains, at Asn136, 156, and 193, whereas other mammalian ATIIIs have four, additional chain being at Asn97; this is replaced by Asp in porcine ATIII. In the case of human ATIII, the chains are at Asn96, 135, 155, and 192. The heparin-binding affinities of human and porcine ATIIIs were compared using an immobilized heparin column. Porcine ATIII eluted from the column with a peak at an NaCl concentration of 924 mM while human ATIII eluted at 838 mM NaCl. Neuraminidase treatment of each ATIII enhanced the heparin-affinity to the same extent. These results suggest that in spite of the high degree of amino acid sequence identity between porcine and human ATIIIs (91% identical), porcine ATIII has a higher heparin-binding affinity than human, because it lacks a carbohydrate chain at Asp97.
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PMID:Amino acid sequence of porcine antithrombin III. 789 48

Experiments were performed to define the best isolation method for isolating Chrysaora fishing tentacle nematocyst organelles in order to minimize non-nematocyst contaminating proteins and proteases and stabilize crude nematocyst venom lethal activity. Techniques employed to disrupt the tentacles included autolysis, homogenization, or digestion using either trypsin or collagenase. Sephacryl-200 gel-filtration chromatography separated two lethal fractions. An immobilized serine protease inhibitor column, m-aminophenyl boronic acid acrylic beads, which reversibly bound one of the two lethal factors, was used in the second and third purification steps. By this means, a 105,000 mol wt. protein was purified, as judged by silver stained SDS-polyacrylamide gels. Lethal activity was inhibited by exposure to the serine protease inhibitor, L-1 chloro 3[4-tosylamido]-7-amino-2-heptanone-HCl, after purification. Although this lethal factor has some characteristics of a serine protease, it is not proteolytically active.
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PMID:Sea nettle (Chrysaora quinquecirrha) lethal factor: purification by recycling on m-aminophenyl boronic acid acrylic beads. 791 86

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


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