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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An extracellular 1,3-specific lipase with molecular weight of 35.5 kDa and an isoelectric point of 4.4 from Aspergillus niger has been purified 50-fold by pH precipitation followed by a series of chromatographic steps with an overall yield of 10%. The enzyme was homogeneous as judged by denaturing polyacrylamide gel electrophoresis and size-exclusion fast-performance liquid chromatography. It contained 2.8% sugar which was completely removed by endoglycosidase F treatment, and the deglycosylated enzyme retained full activity. The native lipase showed optimal activity between temperatures 35 and 55 degrees C and pH 5.0 and 6.0. The amino acid composition and the N-terminal sequence were found to be different from lipases previously purified from A. niger. The enzyme was resistant to trypsin, chymotrypsin, endoprotease Glu-C, thrombin, and papain under native conditions but was susceptible to cleavage by the same proteases when heat-denatured.
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PMID:Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger. 1090 84

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.
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PMID:Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. 1101 86

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
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PMID:Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides. 1137 81

An inhibitor of factor Xa (FXa) was isolated from the nymphs of the camel tick Hyalomma dromedarii by a combination of chromatography on DEAE-cellulose and Sephacryl S-300 columns. The isolated nymphal FXa inhibitor turned out to be a homogenous preparation of a single polypeptide chain (15 kDa) as judged by both the native and denatured SDS-PAGE. Its pI value ranged from 7.7 to 7.9. The inhibitor is a potent anticoagulant since it prolonged both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma in a concentration-dependent manner. Its activity was threefold lower toward thrombin than FXa, but it did not inhibit any of the proteases; trypsin, alpha-chymotrypsin, papain, pepsin and subtilisin. The inhibitor binds at two sites on FXa uncompetitively with an inhibition constant (K(i)) value of 134 nM.
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PMID:Factor Xa (FXa) inhibitor from the nymphs of the camel tick Hyalomma dromedarii. 1169 27

Serpins are responsible for regulating a variety of proteolytic processes through a unique irreversible suicide substrate mechanism. To discover novel genes regulated by transforming growth factor-beta1 (TGF-beta 1), we performed differential display reverse transcriptase-PCR analysis of NRP-152 rat prostatic epithelial cells and cloned a novel rat serpin that is transcriptionally down-regulated by TGF-beta and hence named trespin (TGF-beta-repressible serine proteinase inhibitor (trespin). Trespin is a 397-amino acid member of the ov-serpin clade with a calculated molecular mass of 45.2 kDa and 72% amino acid sequence homology to human bomapin; however, trespin exhibits different tissue expression, cellular localization, and proteinase specificity compared with bomapin. Trespin mRNA is expressed in many tissues, including brain, heart, kidney, liver, lung, prostate, skin, spleen, and stomach. FLAG-trespin expressed in HEK293 cells is localized predominantly in the cytoplasm and is not constitutively secreted. The presence of an arginine at the P1 position of trespin's reactive site loop suggests that trespin inhibits trypsin-like proteinases. Accordingly, in vitro transcribed and translated trespin forms detergent-stable and thermostable complexes with plasmin and elastase but not subtilisin A, trypsin, chymotrypsin, thrombin, or papain. Trespin interacts with plasmin at a near 1:1 stoichiometry, and immunopurified mammal-expressed trespin inhibits plasmin in a dose-dependent manner. These data suggest that trespin is a novel and functional member of the rat ov-serpin family.
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PMID:Identification and characterization of a novel rat ov-serpin family member, trespin. 1198 14

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.
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PMID:Novel secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities. 1243 89

Beyond the well-known antibacterial and beta-lactamase enzyme inhibiting properties of the different beta-lactam antibiotics and their modified derivatives there are a number of beta-lactam (azetidine-2-one) compounds possessing different biological activities. Most of them have been shown to inhibit a variety of cysteine or serine protease enzymes comprising plants, viruses, protozoa, bacteria and mammalian species. This review article covers the biologically active beta-lactam compounds but beta-lactam antibiotics or beta-lactamase inhibitors, and including a few chemically related gamma- or delta-lactams or azetidines presumably having the same site and mechanism of action. These include viral protease inhibitors, protozoan enzymes (e.g. cruzain, falcipain), plant enzymes (papain). Of the mammal enzymes the most important ones are cholesterol absorption inhibitors and human leucocyte (neutrophyle) elastase inhibitors, but mention must be made on thrombin, chymotrypsin, cathepsin, prostata specific antigen and tumor necrosis factor inhibitors, as potential future remedies of cardio vascular and inflammatory diseases.
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PMID:[Reactions of derivatives of beta-lactam antibiotics with non-antibacterial biological activity]. 1281 42

The accumulation of extracellular matrix components such as proteoglycans is a hallmark of an atherosclerotic lesion. A large heparan sulfate proteoglycan, perlecan, dramatically increases in the advanced lesion, and vascular smooth muscle cells are the cell type responsible for the accumulation. In this study, we investigated the effects of thrombin on the proteoglycan synthesis in cultured human coronary smooth muscle cells to determine the interrelationship between the accumulation of proteoglycans and the procoagulant state of blood in atherosclerosis. The cells were metabolically labeled with [(35)S]sulfate or (35)S-labeled amino acids in the presence of thrombin, and the labeled proteoglycans were characterized by Sepharose CL-4B molecular sieve chromatography and DEAE-Sephacel ion-exchange chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was determined by SDS-polyacrylamide gel electrophoresis before and after digestion with chondroitinase ABC or papain. The results indicate that thrombin increases the cell layer-associated heparan sulfate proteoglycan with a core protein size of approximately 400 kDa without any change in the length of the glycosaminoglycan chains when the cell density is high. The heparan sulfate proteoglycan was identified as perlecan by Western blot analysis. In addition, quantitative reverse transcription-polymerase chain reaction showed that thrombin elevated the steady-state level of perlecan mRNA but not that of versican, decorin, and syndecan-1 mRNAs, although that of biglycan mRNA was moderately elevated. Furthermore, the percentage of disaccharide units that compose perlecan heparan sulfate chains remained unaffected by thrombin. Therefore, it is suggested that thrombin induces the perlecan core protein synthesis without influencing the formation of the heparan sulfate chains in human coronary smooth muscle cells at a high cell density. The regulation of proteoglycan synthesis by thrombin may be involved in the accumulation of perlecan in advanced lesions of atherosclerosis.
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PMID:Induction of synthesis of a large heparan sulfate proteoglycan, perlecan, by thrombin in cultured human coronary smooth muscle cells. 1571 25

The interaction of inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated. The following enzymes were tested: serine proteases (trypsin, alpha-chymotrypsin, plasmin, thrombin, kallikrein), cysteine protease (papain) and aspartic protease (pepsin). Inhibitor VJ interacted only with trypsin and 6-chymotrypsin. Kinetic and thermodynamics parameters of intermolecular complexes formation were determined: KD = 7,38 x 10(-8) M and 9,93 x 10(-7) M for pairs InhVJ/trypsin and InhVJ/alpha-chymotrypsin, respectively.
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PMID:[Interaction investigation of trypsin inhibitor from sea anemone Radianthus macrodactylus with proteases]. 1728 51

A serine protease inhibitor with a molecular mass of 6106 +/- 2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed alpha/beta or alpha + beta polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 x 10(-9) M) and alpha-chymotrypsin (Ki 2.17 x 10(-8) M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.
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PMID:[A serine protease inhibitor from the anemone Radianthus macrodactylus: isolation and physicochemical characteristics]. 1788 36


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