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:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

The effects of 25 to 75 volume-% ethanol on conformation of human serum alpha1-acid glycoprotein, human serum alpha1-antitrypsin, pancreatic deoxyribonuclease I, porcine pepsinogen, the "Kunitz" trypsin inhibitor from soybeans, and oxidized as well as reduced and S-carboxymethylated ribonucleases were tested by the circular dichroism (CD) probe. It was found that 25 volume-% ethanol had a slight effect, whereas 50--75 vol.-% alcohol significantly altered the conformation. The tertiary structure was perturbed and the polypeptide main chain was reorganized into new conformations of higher helix and beta-structure contents than in the native state. Comparison of the various proteins showed that the degree of reorganization depended chiefly on the cross-linking of the main chain by disulfide bridges. While the unfolded ribonucleases were refolded by 25 vol.-% ethanol into ordered conformations, the native ribonuclease and alpha1-antitrypsin was more sensitive to 25 vol.-% ethanol than the conformation of alpha1-acid glycoprotein, pepsinogen, and soybean trypsin inhibitor. Almost complete restoration of the native conformation was achieved by diluting the alcohol-containing solutions with water or by dialysis against water or buffer solutions. However, the renaturation depended on the time of contact with alcohol and on the temperature at which the alcohol-containing solutions were kept.
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PMID:Circular dichroism studies on the effects of ethanol on the conformation of alpha1-acid glycoprotein, alpha1-antitrypsin, deoxyribonuclease, pepsinogen, soybean trypsin inhibitor and unfolded ribonucleases. 30 38

An enzymic method is described which allows the isolation under comparable conditions of crypt and villus cells from rat jejunum with normal morphologic appearance and high metabolic activity when compared with previous preparations. The method is based on a differential scraping of short lengths of everted small intestine to yield two villus cell fractions and a gut wall residue. The scrapings and the gut tube are incubated for the same length of time in a HEPES-buffered modified Hanks' balanced salt solution containing hyaluronidase, DNase, and soybean trypsin inhibitor. The cells of the crypt region are recovered by a further scraping of the digested gut wall. Cells from all fractions are dispersed by gentle agitation, washed, and harvested by centrifugation. The final crypt and villus cells are 95--99% viable by dye exclusion and exhibit 5--20% cross-contamination on the basis of differential marker enzymes. The isolated crypt and villus cells prepared by the new procedure are suitable for comparative studies of metabolic activity in the absence of chelation-induced structural and metabolic abnormalities.
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PMID:Improved isolation of villus and crypt cells from rat small intestinal mucosa. 49 96

Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.
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PMID:The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels. 363 34

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

The use of rat testicular seminiferous tubular basement membrane (STBM) segments as a model substratum for the in vitro maintenance of tumor cells dissociated from a transplantable pancreatic acinar rat carcinoma (J. K. Reddy and M. S. Rao, Science (Wash. DC), 198: 78-80, 1977) is described. Ultrastructurally pure, hollow tubular segments of STBM were prepared by mechanical disaggregation, DNase digestion, and deoxycholate treatment. Dissociated pancreatic acinar carcinoma cells adhered readily to STBM segments within 1 to 6 hr, and these STBM-tumor cell aggregates were maintained for up to 7 days in serum-free chemically defined medium supplemented with hydrocortisone, insulin, vitamin C, and soybean trypsin inhibitor. The tumor cells formed acinar-like clusters and displayed intercellular junctions and polarization of secretory granules toward the center of these clusters. By 4 days, virtually all cells of this acinar carcinoma maintained on STBM in supplemented chemically defined medium contained numerous secretory granules. Cell replication, as determined by [3H]thymidine autoradiography, ceased within 18 hr of attachment of neoplastic cells to STBM; however, all cells incorporated [3H]leucine as evidenced by light and electron microscopic autoradiography. In addition, two-dimensional analysis and fluorography of newly synthesized secretory proteins discharged by these cells in response to carbamylcholine revealed the presence of Mr 24,000 protein and 19 other secretory proteins characteristic of this tumor (L. J. Hansen, M. K. Reddy, and J. K. Reddy, Proc. Natl. Acad. Sci. USA, 80: 4379-4383, 1983). The culture system utilizing STBM and supplemented chemically defined medium should allow investigation of the effects of a variety of factors on morphogenesis, cytodifferentiation, and gene expression in pancreatic acinar tumors.
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PMID:Differentiation of pancreatic acinar carcinoma cells cultured on rat testicular seminiferous tubular basement membranes. 648 90

The presence of glucocorticoid-binding macromolecular receptors was demonstrated in the Na2MO4 (10 mM)-stabilized gill cytosol of the American eel, Anguilla rostrata and in that of the trout, Salmo gairdneri. In all experiments, tritiated triamcinolone acetonide [( 3H]TA) was used as ligand. In the eel, the steroid was bound with a KD of 2.84 +/- 0.4 nM and an Nmax of 188 +/- 34 fmol/mg protein. The binding parameters for the trout cytosol were KD = 1.43 +/- 0.13 nM; Nmax = 271 +/- 113 fmol/mg protein. Competition studies with [3H]TA-labeled eel gill cytosol and radioinert steroids gave the following binding hierarchy: TA greater than dexamethasone greater than cortisol greater than 11-deoxycortisol greater than 21-deoxycortisol. Aldosterone, estrogens, or androgens did not complete. The eel gill receptor was deactivated by prior treatment with trypsin or mersalyl. RNase was without effect, but DNase degraded the receptor except when used in the presence of trypsin inhibitor. The eel gill TA-receptor complex sedimented on a linear (10-30%) sucrose gradient with a single peak at 7.0 S or 3.5 S, in hypotonic or hypertonic (0.4 M KCl) gradients, respectively. The eel ligand-receptor complex did not bind, following heat activation, to DNA-cellulose or phospho-cellulose, though it bound to DEAE-cellulose. In this respect, it behaved similarly to the eel intestinal mucosal TA-receptor complex, described previously. The initiation of dissociation of the eel receptor-[3H]TA complex with excess TA yielded pseudo-first-order dissociation kinetics (k-1 at 0 degree C: 2.39 X 10(-5) S-1), while the association kinetics of the receptor with the ligand was of second order (k + 1: 2.51 X 10(4) M-1 S-1). Sepharose column chromatography indicated a molecular weight of 334,690 Da. Calculation of the Stokes radius gave a value of 84.5 A and the frictional ratio, calculated from the molecular weight, was 1.84. From these data it was concluded that the gills of these two euryhaline teleosts contain tetrapod-type glucocorticoid receptors. These studies are the first to demonstrate these steroid recognition molecules in fish gill. The presence of receptors in the fish gill tissue are in agreement with the physiological action of corticosteroids in allowing adaptation of the animals to habitats of different salinity.
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PMID:Glucocorticoid receptors in the gill tissue of fish. 671 55

Isoelectric focusing of culture supernatants from Vibrio cholerae El Tor 1621 and high protease-producing mutant strain 1621 hip revealed the presence of three different types of extracellular protease. Type I protease was the major activity in the wild-type strain and was inhibited by phenylmethylsulfonyl fluoride and by the lima bean trypsin inhibitor. Type II protease was present in the wild type and was the major activity in the high protease-producing mutant. It was resistant to inhibitors of metalloproteases and serine proteases. Two peaks of type II protease differed by 1.2 pI units in isoelectric point and by 1,500 in molecular weight. Type II protease had broad specificity, acted as a mucinase, and caused degradation of some other V. cholerae extracellular proteins, including DNase and cholera toxin. Type III protease was EDTA inhibitable and was detected only in the high protease producer. Possible roles of extracellular proteases as virulence factors in cholera pathogenesis are discussed.
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PMID:Biochemical characterization of extracellular proteases from Vibrio cholerae. 675 27

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.
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PMID:Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment. 816 20

A protease designated pleureryn, with an N-terminal sequence dissimilar from previously reported mushroom metalloendopeptidases and showing only limited resemblance to aspartic proteinases, albeit considerable homology to DNA replication licensing factor, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on CM-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel Blue gel and CM-Sepharose. It demonstrated a single band with a molecular weight of 11.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pleureryn demonstrated a protease activity of 9364 U/mg toward casein. It exhibited a pH optimum of 5.0 and a temperature optimum of 45 degrees C, with substantial activity remaining at high temperatures and pH 4 and 12. The activity of the protease was adversely affected by pepstatin A, indicating that it is an aspartic protease. PMSF, trypsin inhibitor, and EDTA exerted no striking effect, suggesting that it is neither a serine protease nor a metalloprotease. It inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 20 nM. Pleureryn also exhibited some inhibitory activity against HIV-1 reverse transcriptase, reminiscent of a suppressive action of HIV-1 protease on its homologous reverse transcriptase but was devoid of ribonuclease, deoxyribonuclease, and antifungal activities.
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PMID:Pleureryn, a novel protease from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. 1172 12


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