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)

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.
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PMID:Purification and characterization of an enterotoxin from Bacteroides fragilis. 154 60

Ten isolates of Clostridium difficile expressing different degrees of toxigenicity and virulence in an animal model were assayed for the production of proteolytic enzymes by various methods. All strains demonstrated some activity in one or more of the assay systems. There was no direct correlation between toxigenic status and enzyme production. However, those strains known to be highly virulent in a hamster model were the most proteolytic. The most commonly detected enzyme was cell associated, and its substrate specificity suggested it was a trypsin-like enzyme. Initial purification of the enzyme from strain VPI 10463 gave a 10% yield with a 14-fold increase in purity. Inhibition studies on this preparation indicated that the enzyme was a thiol protease. The enzyme has pH and temperature optima of 7.5 and 37 degrees C, respectively. These characteristics suggest that the enzyme is more related to clostripain, the thiol clostridio-peptidase of C. histolyticum, than to trypsin. Whilst the role of this enzyme remains unclear, it is possible that it may be a contributory factor in the virulence of the organism as described for other clostridial infections.
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PMID:Proteolytic activity of Clostridium difficile. 158 79

Undenatured rubella virus envelope glycoproteins released by Tween-ether and trypsin treatment have been separated by Fast Protein Liquid Chromatography. Red cell adsorption located the haemagglutinin on VPI and its 13K cleavage product.
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PMID:Rubella virus haemagglutinin: association with a single virion glycoprotein. 399 17

Toxin preparations were obtained by growing Clostridium difficile VPI strain 10463 in 2-liter brain heart infusion dialysis flasks at 37 degrees C for 3 days. The initial step of the purification scheme involved ultrafiltration through an XM-100 membrane filter. Two toxic activities, designated toxins A and B, were separated by ion-exchange chromatography on DEAE-NaCl gradients. Toxin A was purified to homogeneity by an acetic acid precipitation at pH 5.5. Other separation techniques, including CM Sepharose CL-6B, (NH4)2SO4 and acetic acid precipitations, and hydrophobic interaction chromatography, were examined in attempts to further purify toxin B. Although these methods failed to increase the specific activity of toxin B, they provided additional evidence that the two toxins are distinct molecules. The toxins are acid and heat labile and are inactivated by trypsin and chymotrypsin, but not by amylase. The molecular weight of toxin A, as estimated by gel filtration and gradient polyacrylamide electrophoresis, ranged from 440,000 to 500,000. The estimated molecular weight of toxin B was 360,000 to 470,000.
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PMID:Purification and characterization of toxins A and B of Clostridium difficile. 706 10