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)

Sarcosine oxidase from Corynebacterium sp. U-96 is inhibited by iodoacetamide (IAM) and the inhibition is prevented by the substrate analog, sodium acetate. To elucidate the mechanism of inhibition of the enzyme by IAM, we determined the amino acid sequences around the IAM-reactive cysteine residues, and the effects of the modification on the enzyme activity and the oxidation-reduction of the FAD moieties of the enzyme. The enzyme was specifically labeled with [14C]IAM, and the labeled subunit B was digested with trypsin and chymotrypsin. The HPLC profiles of the proteolytic digests showed mainly two radioactive peaks. The 14C-labeled peptides were purified, and their N-terminal sequences were determined to be Cys-Gly-Thr-Pro-Gly-Ala-Gly-Tyr (TC-1) and Ala-Gly-Ile-Ala-Cys-Xaa-Asp-Xaa-Val-Ala(-)- (TC-2). Peptide TC-2 contains a covalent FAD-binding sequence [Asx-His-Val-Ala; Shiga et al. (1983) Biochem. Int., 6, 737]. [14C]IAM-incorporation into the TC-1 sequence was strongly inhibited by sodium acetate. The N-terminal amino acid sequence of the CNBr fragment containing the TC-1 sequence (65 residues) was determined. According to the secondary structure predictions, Gly-Thr-Pro-Gly-Ala-Gly of the TC-1 sequence is located between the beta sheet and alpha helix of the sequence, indicating the presence of an AMP-binding site in the TC-1 region. The activity of the enzyme treated with IAM in the presence and absence of sodium acetate was not inhibited by sodium sulfite, which is known to react specifically with covalent FAD.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cysteine residues in the active site of Corynebacterium sarcosine oxidase. 193 12

Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.
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PMID:Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant. 200 12

According to the literature and the authors' data in patients who died of dysentery Shigellae are found seldom because of postmortem shedding of superficial colonic epithelium infected by them. Shigella adhesion and invasion into the colonocytes are regularly found in the colon biopsies. As shown recently in experiments, Shigella outer membrane proteins forming "contact haemolysin" ("virulence plasmid" product) are responsible for their invasion. In the small intestine this cytotoxin is destroyed by trypsin, therefore Shigella invasion takes place in the large intestine where it also lyses vacuole membranes around the bacteria in colonocytes. Widespread cytopathic alterations of the epithelium with a damage to ribosome and protein synthesis, disturbance of vascular permeability and fluid hypersecretion in the small intestine result from Shiga-like enterotoxin-cytotoxin. Extent of the inflammatory leukocyte response depends on the degree of Shigella invasion and multiplication and the destruction of the epithelium. Damages to the endothelium and blood coagulation system resulting occasionally in the infectious-toxic shock, are associated with Shigella destruction by leukocytes and absorption of lipopolysaccharide endotoxin released by them. Interepithelial lymphocytes especially those containing lysosome-like granules (similar to the blood "natural killers") play an important role in the response to Shigella.
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PMID:[Current views on the pathomorphology and pathogenesis of dysentery]. 228 80

The neurologic symptoms in human shigellosis have often been attributed to Shiga toxin, although its exact role has not been determined. By use of a [3H] thymidine-labeled HeLa cell assay, cytotoxic activity was demonstrated in stool but not cerebrospinal fluid or serum from five patients with shigellosis presenting with seizures or encephalopathy. Bacterial isolates produced 16.0-88.2 CD50 (50% cytotoxic dose) of cytotoxin/mg of protein. The toxin activity in stool and the cytotoxic activity of the isolates were not neutralized by antiserum to purified Shiga toxin. DNA hybridization studies showed that Shigella isolates from these patients lacked the structural genes for Shiga toxin. The cytotoxin produced was also distinct from Shiga-like toxins I and II. Sonicates of the Shigella strains injected intraperitoneally into mice caused lethargy and lethality. The toxin activity was heat-labile and sensitive to trypsin, indicating that its active component is protein. Ultrafiltration and gel filtration chromatography showed a molecular mass of 100-125 kDa. Thus Shiga toxin production is not essential for the development of neurologic manifestations of shigellosis; other toxic products may play a role.
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PMID:The association of Shiga toxin and other cytotoxins with the neurologic manifestations of shigellosis. 232 46

The primary structures of the A and B subunits of Shiga toxin and of Shiga-like toxin I (VT1), isolated from the culture supernatants of Shigella dysenteriae 1 and Escherichia coli O157:H7, respectively, were analyzed by Edman degradation of intact proteins and peptides in their digests with trypsin or Achromobacter protease I and also by fast atom bombardment mass spectrometry of the digests. The results indicated that the A and B subunits of Shiga toxin and Shiga-like toxin I have the same primary structures. The identity of their primary structures was confirmed by determining the nucleotide sequence of the gene encoding Shiga-like toxin I cloned from a Shiga-like toxin I converting phage. This nucleotide sequence was different from that reported by Jackson et al. (Microbial Pathogenesis 1987; 2: 147-153), by Calderwood et al. (Proc Natl Acad Sci USA 1987; 84: 4364-8) and by Grandis et al. (J Bacteriol 1987; 169: 4313-9) in one base at position 231, which was found to be adenine instead of thymine, which they reported. The amino acid residue at position 45 from the N-terminus of the A subunit of Shiga-like toxin I deduced from the nucleotide sequence determined in this study is threonine, which corresponds with that found by amino acid sequencing, whereas from previous reports by other investigators it is serine. Edman degradation of the intact A subunit of Shiga toxin indicated that the A subunit was nicked between Ala253 and Ser254 to form A1 and A2 fragments linked by a disulfide bond.
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PMID:Identity of molecular structure of Shiga-like toxin I (VT1) from Escherichia coli O157:H7 with that of Shiga toxin. 307 Feb 68

The pathogenesis of the wide-spectrum human disease caused by Salmonella species is poorly understood. Cytotoxin production by other enteric pathogens has been increasingly investigated recently, and data are accumulating regarding the role of cytotoxins in enteric infections and hemolytic uremic syndrome. We studied the cytotoxic activity of 131 Salmonella strains of the major serotypes, including 94 strains of Salmonella enteritidis, 12 strains of Salmonella typhi, and 25 strains of Salmonella choleraesuis. Cytotoxicity was quantitatively determined in sonic extracts by a [3H]thymidine-labeled HeLa cell assay. All Salmonella strains examined showed some degree of cytotoxic activity. The geometric means +/- standard deviations of the amounts of cytotoxin produced (50% cytotoxic dose per milligram of bacterial protein) were 27 +/- 2 for S. typhi, 65 +/- 2 for S. enteritidis, and 117 +/- 2 for S. choleraesuis. Analysis of variance showed that the differences in cytotoxin production by the three species were significant (P less than 0.001). No significant differences were found between stool isolates and invasive strains of the same species. Neutralization studies showed that the cytotoxins produced by all Salmonella strains were immunologically distinct from Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli. DNA hybridization studies with DNA probes for Shiga-like toxins of types I and II showed no hybridization. In each species the cytotoxin was heat labile and sensitive to trypsin treatment, which indicated that its active component was probably protein in nature. Upon ultrafiltration with Amicon membranes and gel filtration chromatography, cytotoxic activity was found in the molecular weight range of 56,000 to 78,000. Our findings indicate that salmonellae produce cytotoxin(s) that may play a role in the manifestations of the various species.
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PMID:Quantitative analysis and partial characterization of cytotoxin production by Salmonella strains. 318 72

The A and B subunits of Shiga toxin were isolated by high performance liquid chromatography and their physicochemical properties were examined. The A subunit of Shiga toxin purified from culture supernatant was not nicked, but it could be nicked in vitro by trypsin. The isoelectric points of the A and B subunits were determined to be 8.2 and 5.8, respectively. Amino acid compositions of the two subunits were also determined. The isolated A and B subunits were reconstituted to form active holotoxin which showed lethal activity to mice which was similar to that of native Shiga toxin.
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PMID:Physicochemical characterization of A and B subunits of Shiga toxin and reconstitution of holotoxin from isolated subunits. 330 22

Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA. By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I. The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E. coli. Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid. As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA. Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase. Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation. We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site.
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PMID:Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. 335 83

Campylobacter jejuni is an important diarrheal pathogen worldwide; the mechanisms by which it causes disease remain unclear. Because of its association with inflammatory diarrhea, we postulated that C. jejuni might produce a cytotoxin similar to that produced by Shigella sp., enterohemorrhagic Escherichia coli O157, or Clostridium difficile. Filtrates of 12 polymyxin-treated isolates of C. jejuni were placed on HeLa cells (sensitive to Shiga toxin cytotoxicity) and Chinese hamster ovary (CHO) cells. Of 12 isolates of C. jejuni tested, 5 killed 50% of the cells at greater than or equal to 1:4 dilutions of filtered suspensions of 10(9) bacteria per ml; killing was similar in HeLa and CHO cells (the CHO cells being insensitive to Shiga cytotoxin). One isolate produced a titer of 1:32 to 1:128. The relative potency in HeLa cells was comparable to that of E. coli strains that produce intermediate amounts of Shiga-like toxin. The other seven strains showed no cytotoxic effect, nor did the control diluents, polymyxin B, or supernatants of C. jejuni not treated with polymyxin B. Sonication also released active cytotoxin, but slightly less well than did polymyxin. The cytotoxic effect was dose dependent. Concentration of the C. jejuni in suspension by 10-fold before treatment with polymyxin B resulted in a 10-fold increase in the 50% cytotoxic dose. The cytotoxin effect was not neutralized by Shiga toxin immune serum against either Shiga-like toxin I or II or by anti-Clostridium difficile antiserum. The C jejuni cytotoxin was partially labile to trypsin (0.25%) and to heating to greater than or equal to 60 degrees C. Cytotoxicity was retained in Scientific Products dialysis tubing D1615-1 (Mr cutoff, 12,000 to 14,000). Some isolates of C. jejuni release a substance lethal to HeLa or CHO cells in vitro that is distinct from Shiga-like or Clostridium difficile toxin. This cytotoxin may contribute to the colonic mucosal invasive process that characterizes C. jejuni enteritis.
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PMID:Production of a unique cytotoxin by Campylobacter jejuni. 365 87

Cleavage of Shiga toxin A-fragment at a highly trypsin-sensitive site increases its enzymatic activity. To investigate the role of this cleavage site in intoxication of cells, we studied the routing, cleavage, and toxicity of mutant toxin where the trypsin-sensitive site had been eliminated. Ultrastructural analysis of toxin tagged with horseradish peroxidase demonstrated that wild-type and mutant toxins were transported from endosomes to the trans-Golgi network and further through the Golgi cisterns to the endoplasmic reticulum. Wild-type toxin was much more efficient than the mutants in provoking rapid intoxication, but after prolonged incubation time also mutants were highly toxic. The cells were able to cleave both wild-type Shiga toxin and the mutants, but the cellular location for cleavage appears to differ. Wild-type toxin was cleaved in the presence of brefeldin A, which disrupts the Golgi cisterns. This indicates that the cleavage occurs in the endosomes or in the trans-Golgi network. In contrast, the mutant Shiga-His (R248H/R251H) was not cleaved in the presence of brefeldin A, indicating that the cleavage can occur only after the toxin has left the trans-Golgi network. In vitro experiments showed that the cytosolic enzyme calpain is able to cleave Shiga-His, and results from in vivo experiments are consistent with the possibility that cleavage is carried out by calpain after the mutant A-fragment has reached the cytosol.
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PMID:Role of processing and intracellular transport for optimal toxicity of Shiga toxin and toxin mutants. 773 76


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