Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent evidence indicates that toxigenic Clostridium difficile strains are a major cause of antimicrobial-associated ileocecitis in laboratory animals and pseudomembranous colitis in humans. C. difficile ATCC 9689 was cultivated in a synthetic medium to which 3% ultrafiltrated proteose peptone was added. Purification of the toxin from broth filtrate was accomplished through ultrafiltration (100,000 nominal-molecular-weight-limit membrane), precipitation with 75% (NH4)2SO4, and chromatographic separation using Bio-Gel A 5m followed by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-25 column. The purified toxin displayed only one band on polyacrylamide gel electrophoresis, and approximately 170 pg was cytopathic for human amnion cells. The isolated toxin was neutralized by Clostridium sordelli antitoxin, heat labile (56 degrees C for 30 min), and inactivated at pH 4 and 9; it had an isoelectric point of 5.0, increased vascular permeability in rabbits, and caused ileocecitis in hamsters when injected intracecally. Treatment of the toxin with trypsin, chymotrypsin, pronase, amylase, or ethylmercurithiosalicylate caused inactivation, whereas lipase had no effect. By gel filtration, its molecular weight was estimated as 530,000. Upon reduction and denaturation, the toxin dissociated into 185,000- and 50,000-molecular-weight components, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive dissociation yielded only the 50,000-molecular-weight component. The toxin appears to be protoplasmic and is released into the surrounding environment upon autolysis of the cells. Attempts to correlate specific enzymatic activity with the toxin have been unsuccessful. These studies will help delineate the role of C. difficile toxin in antimicrobial-associated colitis and diarrhea.
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PMID:Purification and characterization of Clostridium difficile toxin. 47 34

A trypsin-sensitive, heat-labile cytotoxin was purified from the supernatant of a culture of Clostridium difficile by a procedure that included ultrafiltration, precipitation with (NH4)2SO4, gel filtration, and ion-exchange chromatography. The procedure resulted in recovery of 20% of the cytotoxin and an estimated 1,500-fold increase in cytotoxic activity. The minimal amount of protein required to give an actinomorphic response in WI-38 cell cultures was 1.4 ng/ml. The estimated molecular weight of the cytotoxin is 240,000. A cytotoxin having similar properties was purified from the stool of a patient with antibiotic-associated pseudomembranous colitis by (NH4)2SO4 precipitation and gel filtration chromatography. This procedure resulted in a recovery of 26% of the cytotoxin, a 50-fold increase in cytotoxic activity, and a cytotoxic response with a minimum of 12.1 ng of protein/ml.
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PMID:Partial purification and characterization of a cytotoxin from Clostridium difficile. 54 89

Two toxins were isolated from a toxigenic strain of Clostridium difficile. The toxins were purified by gel filtration and ion-exchange column chromatography to homogeneity as judged from polyacrylamide gel electrophoresis and were designated D-1 and D-2. Toxin D-1 was lethal for mice, increased vascular permeability, and induced fluid accumulation in ligated rabbit ileal loops, and toxin D-2 displayed cytotoxicity in HeLa cells with a minimum of 1 pg of toxin. The molecular weights of toxins D-1 and D-2, as estimated by gel filtration, ranged from 550,000 to 600,000 and from 450,000 to 500,000, respectively. These toxins were heat labile and inactivated by pronase and trypsin. Amino acid analyses of both toxins showed them to be of relatively similar composition. Antisera prepared against purified toxin D-1 neutralized all of the biologic activities of toxin D-1, whereas it did not affect any of the biologic activities of toxin D-2. A sensitive latex-agglutination immunoassay was developed for screening for C. difficile toxin D-1 in patients with pseudomembranous colitis.
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PMID:Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridium difficile. 642 16