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)

Comparative investigations were carried out on the immunofluorescent preparations of cell cultures infected with bovine viruses--rota-, corona-, respiratory-syncytial, parainfluenza-3, adeno-1, and herpes-1--to test various fixatives and the effect of trypsin in raising the sensitivity of the immunofluorescence method. The effect of trypsin was manifested in fixation with formalin, ethanol, methanol, and acetone treated immunofluorescent preparations of cell cultures infected with rota- and adeno-viruses as well as in fixation with acetone of cultures infected with respiratory syncytial virus, parainfluenza-3 virus, and corona virus. Formalin, ethanol, and partly methanol were shown to be unsuitable for the purpose of fixation of cell culture preparations infected with viruses that contained a lipoprotein envelope. It was found that the treatment of immunofluorescent preparations with trypsin following fixation and prior to their treatment with conjugated antisera enhanced considerably the number of fluorescent cells and the intensity of fluorescence itself provided 0.1 per cent trypsin was used for 5 to 10 min at 37 degrees C--for cell culture preparations, and 0.1 per cent trypsin was used for 20 to 30 min at 37 degrees C--for paraffin sections of acetone-fixed tissues.
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PMID:[Enhanced luminescence intensity in immunofluorescent preparations following trypsin treatment]. 354 71

Monoclonal IgG1 antibodies 2C8 and 2F7, derived by immunization of mice with a glycoprotein-enriched fraction of human ovarian adenocarcinoma, recognized a 60 kD glycoprotein in the ovarian tumor but not in normal ovary. Survey of other normal adult tissues by an indirect solid-phase radioimmunoassay (RIA) revealed the presence of the antigen in trace amounts in various normal organs such as small intestine, liver colon and urinary bladder, except in lung where its concentration was as high as in tumors. Among fetal tissues tested, intestine and placenta had the highest activities. By RIA, about 50% of ovarian and colonic tumors had elevated levels of the antigen. All ovarian cyst fluids, both benign as well as malignant, also contained a high level of the antigen. Immunodepletion studies indicated that the antigen was distinct from carcinoembryonic antigen and the ovarian cancer antigens described in our laboratory with other monoclonal antibodies. The antigen bound to Con A-Sepharose and was eluted with 2% alpha-D-mannoside, was soluble in 0.6 M perchloric acid and stable at 100 degrees C for 30 min. The antigenic activity in isolated plasma membrane enriched fractions of ovarian adenocarcinomas was sensitive to trypsin, chymotrypsin or protease treatment but unaffected by neuraminidase, beta-galactosidase, periodate or methanol treatment. By immunoperoxidase staining, the antigen was localized in a variety of human tumors showing widespread distribution.
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PMID:Production and characterization of monoclonal antibody to a 60-kD glycoprotein in ovarian carcinoma. 389 88

In early studies, the bullous pemphigoid antigen (BPA) has been localized extracellularly in the lamina lucida in the basement membrane zone. However, trypsin-dissociated basal cells can be tagged with bullous pemphigoid sera (BPS). By immunofluorescence, BPA appears located at the dermal pole of basal cells (BC). This may indicate that when BC are separated from the underlying matrix molecules, chunks of BPA remain attached to them. In the present study, fresh crude initial suspensions (CIS) of epidermal cells were prepared by trypsin-EDTA dissociation. The cells were smeared and air-dried. Polar fluorescent cells (i.e., BC) amounted to 42% +/- 7%. CIS were then passed through a fluorescence-activated cell sorter (FACS). In the fluorescent-positive fractions selected by FACS, 34% +/- 7% only of the BC were present. FACS-negative cell fractions were smeared on glass slides, air-dried, and restained with BPS + fluorescein isothiocyanate; 66% +/- 10% of BC were present in these fractions. This is evidence that trypsin-isolated BC comprise two subpopulations: one with BPA directly accessible, the other not. Viability tests and tissue culture studies indicated that the FACS-positive cell fractions were not viable. BPA was extracted from CIS, FACS-positive, and FACS-negative fractions and immunoblotted against BPS. Identical blots were found. FACS-negative cell fractions were treated with heparitinase, nitrous acid, methanol-chloroform, or EDTA without modifying the number of reacting cells. When BC were treated with Triton X-100 or permeabilized by successive freezings and thawings, the number of positive cells became comparable to those obtained by air-drying smears. Finally, BPA was localized on the intracellular part of hemidesmosomes of BC by immunoelectron microscopy. To see whether BPA was also present extracellularly, suction blisters were raised in minipigs and BPS injected into the blister cavity. BPA was found attached to all cells of the cellular roof but not to the dermal base of the blisters. When pieces of skin kept overnight in cold trypsin were reacted with BPS, BPA was found on both sides (epidermal and dermal) of the split. It is concluded that BPA has two localizations: one extracellular, essentially labile which accumulates at the dermal-epidermal junction; the other essentially stable which remains on the intracellular part of basal cell hemidesmosomes and which can be detected after permeabilization of the cells.
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PMID:Localization of bullous pemphigoid antigen (BPA) in isolated human keratinocytes. 389 91

Cells of the mouse macrophage-like cell line RAW264 release a dialysable inhibitor of phospholipase activity into their culture medium. This inhibitor can be detected in saline solution, Hanks solution and a variety of tissue culture media in the presence or absence of serum. The inhibitor is stable at 4 degrees C, unaffected by trypsin, nucleases, or boiling, and partially extractable with chloroform/methanol. The release of both arachidonic acid and prostaglandins from mouse macrophages or human monocytes is inhibited by this material. A variety of other cell types release the inhibitor, which is effective against stimulation of arachidonic acid release from cultured macrophages by zymosan, serum, immune complexes and the calcium ionophore A23187.
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PMID:A small phospholipase inhibitory factor released by cultured cell lines. 393 Feb 89

Cerebral neurons in monolayer cultures, subjected to 25 micrograms/ml trypsin, lose after 10 min about 43.5% and 40.5% of the ability to bind 125I-labeled tetanotoxin as measured at 0-4 degrees C and 37 degrees C respectively. These losses are maximal by 30 min and can be prevented by 1.5 mg/ml soybean trypsin inhibitor. Chymotrypsin but not collagenase or hyaluronidase is also effective in reducing binding of toxin to cells. The trypsin-insensitive toxin-binding activity can be further eliminated by treatment with sialidase or by cell extraction with methanol. Fixation of cells with 3.5% paraformaldehyde or 2% glutaraldehyde also results in a marked decrease of 52.4% and 25% respectively in the toxin-cell association. Methanol or sialidase but not trypsin removes the remaining binding activity. About one-third of the lipid-linked and protein-linked sialic acid is removed after sialidase treatment whereas 1% and 9.4% respectively are removed after trypsin treatment. The data are consistent with the possibility that, in addition to a sialic acid component, binding of tetanotoxin to nerve cells is facilitated by a trypsin-removable and formaldehyde-inactivated component. There was no evidence for a polypeptide to substitute gangliosides as receptors for tetanotoxin. On the contrary, solubility in organic solvents and interaction of the extracted products with labeled toxin remain the major proof that gangliosides are the putative receptors for tetanotoxin.
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PMID:Tetanus toxin receptors on nerve cells contain a trypsin-sensitive component. 394 36

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.
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PMID:Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved. 404 Dec 37

Bacillus cereus phospholipase was characterized as a phospholipase C by the analysis of lecithin degradation products by thin-layer and paper chromatography. Methanol in the growth menstruum inhibited completely the synthesis of phospholipase C, whereas the synthesis of lethal toxin and hemolysin were only partially inhibited. Dialysis of preformed B. cereus products against ethyl alcohol and methanol did not inactivate hemolytic, phospholipase C, or lethal activity. The hemolytic and lethal activities of culture filtrates were completely abolished by trypsin, but phospholipase C activity was resistant to inactivation. Lethal and phospholipase C properties of culture filtrates were resistant to inactivation at 45 C, whereas the hemolytic activity was completely destroyed. Lethal, hemolytic, and phospholipase C activities appeared simultaneously in a complex growth menstruum, but the kinetics of synthesis were different in all cases. Resolution of B. cereus filtrates on columns of Sephadex showed that the phospholipase C, hemolysin, and lethal toxin are distinct proteins. Evidence is also presented which suggests a correlation between the synthesis of B. cereus toxin and the period of transition from vegetative growth to sporulation. The activity of each B. cereus product was cation-independent, as opposed to cation-dependency of the phospholipase C and lethal activities of Clostridium perfringens alpha-toxin. Immunological cross-reactivity between the B. cereus products and C. perfringens alpha-toxin was not apparent; indeed, they were shown to be antigenically distinct.
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PMID:Lethal toxin of Bacillus cereus. I. Relationships and nature of toxin, hemolysin, and phospholipase. 429 11

A recent isolate of Mycoplasma fermentans (strain K10, from human leukemic bone marrow) induced a lethal toxicity syndrome in mice. High doses of both viable and inactivated cells were toxic when injected intraperitoneally. Whole lysates and membranes from osmotically shocked cells killed mice, but cytoplasm did not. When membranes were dissolved in detergents and reaggregated by dialysis in the presence of Mg(2+), the lipid-protein complex thus formed was toxic. Lipids extracted from membranes with chloroform-methanol did not kill mice. Protein-rich fractions (obtained by reaggregation plus acetone washes or ammonium sulfate precipitation of dissolved membranes) were also not toxic. No qualitative differences in proteins from three toxic isolates and three nontoxic laboratory strains of M. fermentans were detectable by polyacrylamide gel electrophoresis. The toxic factor contained in reaggregated membranes was heat-stable but sensitive to Pronase, trypsin, and lipase.
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PMID:Toxic membrane fractions from Mycoplasma fermentans. 515 2

Cell walls were isolated by mechanical disruption of mid-log phase cells of Bacillus stearothermophilus NCA 1503-4R grown in Trypticase-yeast extract-fructose medium at 55 C. The cell walls were purified by treatment with sodium dodecyl sulfate (SDS) and incubation with deoxyribonuclease and trypsin. The cell wall peptidoglycan contained glucosamine, muramic acid, alpha, epsilon-diaminopimelic acid, and glutamic acid. Low amounts of glycine, galactosamine, serine, aspartic acid, lysine, and valine were also present. The relative mole ratios of glutamic acid-alpha, epsilon-diaminopimelic acid-glycine-alanine were 1.00:1.26:0.08:1.55. The cell walls were free from ribonucleic acid and deoxyribonucleic acid and contained less than 0.2% chloroform-methanol extractable lipid and 0.09 mumole of phosphorus per mg of cell wall. Teichoic acid was not detected in the cell walls of this organism. Cell walls isolated without treatment with SDS contained 7.5% chloroform-methanol extractable lipid, 0.24 mumole of phosphorus per mg of cell wall, and relatively high concentrations of all amino acids. These results suggest that the extracted lipid is not a cell wall component per se, but a contaminant from the lipoprotein cell membrane.
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PMID:Chemical composition of the cell walls of Bacillus stearothermophilus. 603 16

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased beta/alpha-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (micro-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.
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PMID:DNA-protein binding in interphase chromosomes. 603 46


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