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)

Heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (286C(2)) was purified to homogeneity from pH extracts of fermentor-grown cells by ultrafiltration, (NH(4))(2)SO(4) fractionation, hydrophobic chromatography on norleucine-Sepharose 4B, hydroxylapatite chromatography, and Bio-Gel P-150 filtration. Purified LT preparations exhibited biological activity comparable to that of cholera toxin in four bioassays specific for the two enterotoxins (Y-1 adrenal tumor cells, Chinese hamster ovary cells, pigeon erythrocyte lysates, and skin permeability test). The overall yield of LT protein was 20%, which represented a 500-fold purification over pH extracts. A native molecular weight of 73,000 was determined by gel electrophoresis. The toxin dissociated upon treatment with sodium dodecyl sulfate, pH 7.0, into two components with molecular weights of 44,000 and 30,000. Purified LT preparations were remarkably stable over a wide range of storage conditions, temperatures, and pH's. The biological activity was increased by incubation with trypsin and completely destroyed by pronase and proteinase K, whereas deoxyribonuclease I, ribonuclease, and phospholipase D had no effect. The amino acid composition of purified LT was quite different from that of cholera toxin. Neither carbohydrate nor lipopolysaccharide was present in purified preparations. The purification scheme appeared applicable to LT produced by other human and porcine enterotoxigenic strains, but reflected the amount of LT produced by each strain. These data show that LT and cholera toxin share many common chemical and physical properties, but must be purified by different techniques.
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PMID:Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Escherichia coli. 3 93

A substance inhibitory to protein synthesis was purified from mouse skeletal muscle by gel filtration and ion-exchange chromatography, as well as by centrifugation on sucrose gradients. The molecular weight of the inhibitor, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 71000. The inhibitory activity was insensitive to ribonuclease A, deoxyribonuclease I and phospholipase C. It was sensitive to Pronase treatment but insensitive to heat-treatment and trypsin degradation. The present results, taken together with previous studies, indicate that the site of action of the inhibitor is not on the initiation phase of protein synthesis but rather at a step after the binding of aminoacyl-tRNA to ribosomes. The increased inhibitor activity found in dystrophic muscle is discussed.
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PMID:Studies of a factor from dystrophic mouse muscle inhibitory towards protein synthesis. 74 60

Twenty-two nitrogen-fixing Bacillus azotofixans strains were shown to produce an inhibition zone against themselves in plate assays. The B. azotofixans type strain P3L-5, chosen for further studies, produced inhibition zones against various Bacillus strains and other bacterial genera. This antibacterial substance was also produced in liquid medium and its production was enhanced in semisolid medium (0.4% agar) after 3 to 5 days of incubation. The substance was suggested to be an antibiotic and its preliminary characterization showed resistance to heat (100 degrees C, 15 minutes), to trypsin, pronase, deoxyribonuclease I, ribonuclease A, phospholipase C, ethanol, acetone, and ether, and sensitivity to strong alkali treatment. Its molecular weight was estimated to be between 3500 to 6000. After induction of B. azotofixans P3L-5 with mitomycin C or ultraviolet light, two types of particles were detected in the lysate: one similar to a phage tail and the other, less frequent, similar to a complete bacteriophage. Lysates containing these particles showed a killing effect in some but not all B. azotofixans strains, but neither the other Bacillus species nor Micrococcus were inhibited by these lysates.
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PMID:Production of a bacteriophage, a phage tail-like bacteriocin and an antibiotic by Bacillus azotofixans. 212

In patients with pancreatic cancer deoxyribonuclease I (DNase I) serum levels were compared with those of other known pancreatic enzymes. Serum deoxyribonuclease I, elastase 1, immunoreactive trypsin, amylase and phospholipase A2 were determined in 40 healthy controls, 28 patients with pancreatic cancer, 49 with chronic pancreatitis and 40 with extra-pancreatic diseases. The analysis of variance showed a significant difference among groups for serum DNase I values. However, none of the 3 groups of patients had a mean deoxyribonuclease I value higher than that of the healthy controls. In pancreatic cancer and chronic pancreatitis patients, increases in the 4 pancreatic enzymes values were found in percentages that were higher than those for DNase I. A significant correlation was found between DNase I and phospholipase A2, but not between DNase I and elastase 1, immunoreactive trypsin and amylase serum activities. The findings indicate that deoxyribonuclease I serum determination is an even less satisfactory index of pancreatic malignancy than the other pancreatic enzymes. Rather than expressing pancreatic damage, any variations in this enzyme appear more likely to reflect an aspecific phenomenon.
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PMID:Deoxyribonuclease I serum activity in pancreatic cancer. 235 55

A method is described for the isolation of biliary epithelial cells from rat livers by sequential treatment with EGTA, collagenase-hyaluronidase, trypsin, and deoxyribonuclease I and final separation on a discontinuous Percoll gradient using prekeratin antibodies as an immunohistochemical marker for these cells.
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PMID:The isolation of intrahepatic biliary epithelial cells from normal rat livers. 258 10

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.
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PMID:Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix. 260 69

An inhibitory component that diminishes estrogen receptor (ER) binding to nuclei in vitro is present in cytosol prepared from calf uterus. The inhibitor is heat stable and resistant to enzymatic treatment with trypsin, chymotrypsin, proteinase K, deoxyribonuclease I, or ribonucleases A, T1, and U2. Results of chromatography on DEAE-cellulose and Sephadex G-150 indicate that the factor is a negatively charged macromolecule. Inhibitory activity is sensitive to sequential digestion with chondroitinase ABC, hyaluronidase, and heparinase. Approximately 70% of the inhibitory activity is destroyed by treatment with heparinase alone. Heparitinase destroys only 30% of this activity. Furthermore, the addition of pure hyaluronic acid or chondroitin sulfate to the ER-nuclei binding assay results in little inhibition, whereas addition of heparin inhibits 75% of receptor binding. Overall, these results indicate that glycosaminoglycans, present in bovine uterine cytosol, are capable of inhibiting ER-nuclei interactions. The most potent inhibitory glycosaminoglycan displays heparin-like characteristics.
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PMID:Characterization of a cytosolic inhibitor of calf estrogen receptor binding to nuclei. 330 79

Crystals of cytoplasmic (porcine liver) actin in complex with deoxyribonuclease I (DNAase I) were prepared for structural determination by X-ray-diffraction analysis. The crystallization of porcine liver actin-DNAase I complex is preceded by a brief treatment with immobilized trypsin, whereby a C-terminal tri- or di-peptide including cysteine-374 is removed from the actin without any noticeable degradation of both proteins as judged by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis. Analysis of the crystals obtained does not reveal any differences in the three-dimensional structure of porcine liver actin from its skeletal compartment at up to 0.6 nm resolution. However, in contrast with crystalline skeletal-muscle actin-DNAase I complex, heavy-atom substitution of crystals of porcine liver actin-DNAase I complex could not be achieved with methyl mercuriacetate. Evidence is presented that, in porcine liver actin, the N-terminal cysteine residue is not located at position no. 10, as in skeletal- and smooth-muscle actin, but most probably at position no. 17. Thus, because this site is covered by DNAase I, the cysteine becomes inaccessible to titration with 5,5'-dithiobis-(2-nitrobenzoic acid) after complex-formation with DNAase I.
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PMID:Crystallization of cytoplasmic actin in complex with deoxyribonuclease I. 397 43

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.
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PMID:Antiestrogen binding sites in rat liver nuclei. 406 96

Partially purified ribonuclease H from rat liver nuclei can be inactivated by a soluble fraction from rat intestine; this inactivation is restored by adding trypsin inhibitor, suggesting that the factor is a protease. A preparation has been isolated and purified to homogeneity. The molecular weight of the enzyme was estimated as 28 000 with an optimum pH of 8.0 and an isoelectric point at pH 4.5--4.7. The inactivating and proteolytic activities were observed in parallel throughout the purification procedures. Diisopropylphosphorofluoridate inhibited the protease activity. The protease inactivates deoxyribonuclease I, pyruvate kinase, and aldolase. From experiments with protease modifiers, it seems to be a serine protease of a trypsin-like nature.
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PMID:A protease from rat intestine. 624 51


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