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)

A novel trypsin inhibitor, tentatively named countertrypin, was isolated from mouse plasma in an apparently homogeneous state. Countertrypin is a 53-kDa glycoprotein having about 30% carbohydrate, and did not cross-react immunologically with either mouse alpha 1-antiproteinase (also called alpha 1-proteinase inhibitor or alpha 1-antitrypsin) or contrapsin. Countertrypin had no inhibitory activity against chymotrypsin, pancreatic elastase, neutrophil elastase, thrombin, plasmin, plasma kallikrein, pancreatic kallikrein, clotting factor Xa, or papain. This inhibitory spectrum does not correspond to any of the known plasma proteinase inhibitors that have been well characterized in human or other mammals. NH2-terminal amino acid sequence analysis of the intact molecule and three peptides obtained by CNBr digestion revealed that a total of 93 amino acid residues could be aligned with stretches in human alpha 2-HS glycoprotein, bovine fetuin, and rat pp63 (rat fetuin). Human alpha 2-HS glycoprotein and bovine fetuin prepared without use of ethanol inhibited trypsin and pancreatic and neutrophil elastases. These results indicate that mouse countertrypin is a new member of the mammalian fetuin family, which possibly has the trypsin-inhibiting activity in common.
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PMID:Isolation and characterization of mouse countertrypin, a new trypsin inhibitor belonging to the mammalian fetuin family. 768 30

A cDNA coding for the E isoform of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) was isolated by oligonucleotide hybridization following immunochemical screening of the rabbit liver cDNA library. The deduced amino acid sequence of the E isoform showed 96.4% identity in 413 residues of the F and S-1 isoforms of rabbit alpha-1-antiproteinase. The N-terminal half of the amino acid residues of the three isoforms was almost identical, but the putative reactive-site loop structure (P8-P'8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. Interaction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombinant protein and trypsin formed a 62 kDa equimolar complex, which gradually became graded to the 37 kDa fragment through several intermediates. The E form also formed a complex of a similar size with elastase and became degraded to the 31 kDa fragment. Several proteinases which cleaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain and ficin. Other proteinases, with a stringent substrate specificity, such as thrombin, factor Xa, plasmin, plasma kallikrein and cathepsin G, did not attack the E form. Unlike the F and S-1 forms of rabbit plasma alpha-1-antiproteinase, the recombinant E form did not inhibit the amidolytic and proteolytic activities of trypsin. Neither elastase nor protease V8 was inhibited by the E form. Thus the change in the amino acid residues in the reactive-site loop, probably in the P1 site, is responsible for the loss of inhibitory activity of rabbit alpha-1-antiproteinase E. The novel character of the E form could provide a new insight into the interaction of serpin and proteinases.
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PMID:Rabbit alpha-1-antiproteinase E: a novel recombinant serpin which does not inhibit proteinases. 773 71

Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have produced several recombinant human salivary cystatin SN (reCsnSN) variants. These include a N-terminal-truncated form (aa 17-121), a C-terminal-truncated form (aa 1-102) and two deletion mutants (delta 12-16 and delta 56-60). A large amount of the insoluble fusion protein (approx. 15 mg/l) was produced in each case. These were solubilized with urea and refolded by dialysis. The GST carrier was then cleaved with thrombin and the reCsn variants (except delta 56-60) were purified by anion-exchange chromatography. The CysP inhibitory activities against papain, and bovine and human cathepsin B, and secondary structures of the reCsnSN variants were determined and compared to natural salivary CsnSN. The full-length reCsnSN, the N-truncated and the delta 12-16 variants inhibited the CysP activity of papain and displayed circular dichroism (CD) spectra similar to that of natural CsnSN. On the other hand, the delta 56-60 mutant and the C-truncated variant exhibited very little inhibitory activity towards papain. The CD spectrum of the C-truncated variant indicated a change in the secondary structure (e.g., a decrease in beta-sheet and an increase of an alpha-helical content). Neither, the natural nor the full-length reCsnSN or the delta 12-16 mutant exhibited any inhibitory activity towards bovine and human cathepsin B.
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PMID:Biological activities and secondary structures of variant forms of human salivary cystatin SN produced in Escherichia coli. 782 95

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

New data on the mechanism of interaction between proteolytic enzymes and protein inhibitors are presented. Most of known serine proteinase inhibitors bind to proteolytic enzymes through a substrate-like mechanism. A different mechanism is observed in the case of the inhibition of thrombin by hirudin and of papain by cystatin (from egg white) and stephin B. Protopeptides released during activation of some proteinase zymogens may act as efficient enzyme inhibitors. Such zymogens can be considered as covalently bound enzyme-inhibitor complexes.
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PMID:[Proteolytic enzyme inhibitors of protein nature]. 815 78

Tritiated N alpha-acetyl-L-lysine chloromethyl ketone (ALCK) was synthesized on a laboratory scale for use as an active-site-directed affinity label in the fluorographic detection of proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The synthesis involved acetylation of N epsilon-benzyloxycarbonyl-L-lysine chloromethyl ketone with [3H]acetic anhydride just before the removal of the benzyloxycarbonyl group. By this method, [3H]ALCK with a specific activity of 250 mCi/mmol was obtained as a crystal. Trypsin, thrombin, plasmin, papain, and clostripain were inactivated by ALCK according to first-order kinetics. For fluorographic detection of proteases, enzyme samples were allowed to react with [3H]ALCK and then resolved by SDS-PAGE. Proteases that reacted with [3H]ALCK could be detected with a sensitivity equivalent to or higher than that of Coomassie brilliant blue R-250 staining. A trypsin-like protease in Pronase, clostripain as a contaminant in a commercial preparation of Clostridium histolyticum collagenase, and cysteine proteases in Porphyromonas gingivalis could be detected.
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PMID:Synthesis of N alpha-[3H]acetyl-L-lysine chloromethyl ketone and its use in the fluorographic detection of proteases. 825 Feb 26

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
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PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48

Opossum (Didelphis virginiana) serum was fractionated with (NH4)2SO4 and then chromatographed on DEAE-Sepharose and phenyl-Sepharose. Affinity chromatography on a protein A-Sepharose-antibody column removed traces of opossum serum metalloproteinase inhibitors, and resulted in a homogeneous preparation of opossum alpha 1-proteinase inhibitor (alpha 1-PI). The inhibitor is a single-chain glycoprotein (17.7% carbohydrate) with an estimated M(r) = 54,000. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for the open reading frame for opossum alpha 1-PI were isolated. The cDNA inserts contained nucleotide sequences corresponding to the amino-terminal and an internal peptide sequence of opossum alpha 1-PI which had been separately determined by protein sequence analysis. The entire inserts coded for a protein consisting of a 21-residue signal peptide and a 389-residue mature protein. Opossum alpha 1-PI shows 51-58% identity with other mammalian alpha 1-PI amino acid sequences, and the conserved residues expected for a member for the serpin family have been retained. The carbohydrate attachment sites and the reactive site residues (M-S) of opossum alpha 1-PI are identical to those of human alpha 1-PI. Opossum alpha 1-PI formed stable enzyme/inhibitor complexes with trypsin, chymotrypsin, and human neutrophil elastase, but did not react with thrombin or with snake venom serine proteinases. Opossum alpha 1-PI was inactivated by papain or Pseudomonas aeruginosa elastase, and electrophoretic analysis of the reaction products indicated limited proteolysis in the reactive site loop of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Opossum serum alpha 1-proteinase inhibitor: purification, linear sequence, and resistance to inactivation by rattlesnake venom metalloproteinases. 842 60

Different Escherichia coli expression systems were used for expression of cDNA clones encoding the human salivary cysteine proteinase (CysP) inhibitors, cystatins SN and S (CsnSN and CsnS). These included pOTSNco12 that expresses foreign sequences as authentic (nonfusion) proteins, and pGEX-2T that directs the synthesis of foreign polypeptides as fusion proteins with glutathione S-transferase (GST). The pOTS vector produced low levels of recombinant CsnSN (reCsnSN) that was localized in the soluble fraction, but not easily purified. The pGEX vector, on the other hand, produced much higher yields of the fusion protein, GST::CsnSN, that was localized almost entirely in the insoluble protein fraction. Solubilized and refolded GST::CsnSN inhibited the CysP, papain, more efficiently than chicken egg white Csn, indicating that the recombinant product was biologically active and that the GST carrier did not interfere with the biological activity. The pGEX-2T vector was subsequently used for the large-scale production of reCsnSN and reCsnS that were cleaved from the GST by thrombin and purified by DE-52 cellulose chromatography. ReCsnSN inhibited papain almost as efficiently as salivary CsnSN, while the reCsnS showed lower inhibitory activity as compared to both salivary CsnS and reCsnSN.
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PMID:Efficient production of biologically active human salivary cystatins in Escherichia coli. 842 60

Warfarin overdose leads to hypoprothrombinemia and bleeding diathesis. We report here the case of a 47 year old woman who ingested an overdose of a non-steroidal anti-inflammatory drug, sold in Mexico under the name of Wobenzym (R), and containing, according to the manufacturer: pancreatin, bromelin, papain, lipase, amylase, trypsin, alpha chymotrypsin and rutin. She developed skin, urinary and gastrointestinal bleeding and was found to be apparently under the effect of a coumadin overdose, i.e. prolonged prothrombin time, prolonged activated thromboplastin time, and low functional and antigenic levels of prothrombin. A platelet count, and the thrombin, reptilase and bleeding times were normal. All laboratory and clinical abnormalities reverted to normal by using fresh frozen plasma and parenteral vitamin K. In addition, we were able to show that the commercial preparation could prolong the prothrombin time in rabbits and, by high-performance liquid chromatography, a pike consonant with purified coumadin was found in the drug. It is concluded that this drug is probably contaminated by coumadin, and that physicians must be aware of its potential side effects.
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PMID:[Probable coumarin poisoning upon ingestion of an anti-inflammatory agent]. 896 87


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