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)

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.
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PMID:Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation. 630 4

Reaction of rabbit skeletal muscle G-actin at pH 8.5 with fluorescein isothiocyanate (FITC) resulted in incorporation of up to 1.20 mol FITC/mol actin. At pH 8.8, the level of incorporation was raised to 1.98 mol FITC/mol actin. When excited with ultraviolet light, the FITC-actin samples fluoresced strongly with an emission maximum near 517 nm. Tryptic digests of FITC-actin containing about 1.0 mol FITC/mol actin could be separated into a nonfluorescent 33.5 kDa trypsin-resistant core protein and a fluorescent pool of small peptides. Chromatography on DEAE-Bio-Gel or two-dimensional separation on cellulose TLC plates of the peptide pool revealed that FITC was highly selective in the site of its reaction with actin, resulting in a single highly fluorescent peptide after tryptic digestion. NH2-terminal and amino acid analyses demonstrated this peptide to be derived from residues 51 to 62, with Lys-61 proposed as the major FITC-sensitive site on actin. FITC-actin is similar to G-actin in gross conformation; circular dichroism spectra of actin before and after labelling are identical. FITC-actin is also able to interact strongly with deoxyribonuclease I. However, FITC-actin solution viscosities and fluorescence properties are not altered by the addition of KCl or MgCl2. Therefore, either a localized conformational change near Lys-61 or steric hindrance due to the FITC attached to Lys-61 blocks the polymerization of actin.
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PMID:Modification of actin with fluorescein isothiocyanate. 643 49

2-(N-methylanilino)naphthalene-6-sulfonic acid (MANS) binds to G-actin at a single high affinity hydrophobic site (Kd = 41 microM). Salt-induced polymerization of MANS-G-actin results in a general enhancement of sample emission intensities at all wavelengths. At 430 nm, KCl-induced polymerization yields a 2,3-fold enhancement, while MgCl2-induced polymerization gives a 2.0-fold increase. Polymerization of MANS-G-actin in the absence of agitation produces MANS-F-actin samples that have fluorescence polarization values at 430 nm of 0.33. Subsequent mixing or sonication of such MANS-F-actin samples results in a dramatic drop in fluorescence polarization values to 0.14. After cessation of mixing, the polarization values do not recover to their initial levels. Circular polarization of luminescence studies on MANS-actin demonstrate that agitation of MANS-F-actin samples drastically alters emission anisotropy values. 9-Anthroyl choline (9AC) binds to G-actin at a single hydrophobic site (Kd = 68 microM). The fluorescence of 9AC-actin is sensitive to salt-induced polymerization and depends upon the identity of the salt employed. KCl causes a drop in the fluorescence intensity at 490 nm to 70% of the value for 9AC-G-actin, while MgCl2 produces a 30% increase in intensity. Polarization experiments with 9AC-actin produced qualitatively the same results as did those with MANS-actin. Differences in the behaviours of MANS-actin and 9AC-actin in response to polymerization by KCl and MgCl2 and in response to the binding of deoxyribonuclease I suggest that the binding sites on actin for MANS and 9AC do not overlap completely.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluorescence of actin-bound hydrophobic molecules. 662 8

Localization of immunogenic tissue-specific and tissue-non-specific non-histone proteins in thymocyte chromatin has been investigated using antibodies against rat thymus and liver chromatin. After chromatin digestion by DNAase II and subsequent fractionation with 2 mM MgCl2, the Mg2+-soluble fraction interacts with both types of antibodies 5-6 times more effectively than Mg2+-insoluble chromatin. The experiments on chromatin digestion with DNAase I indicate that tissue-specific and tissue-non-specific proteins, reacting with antibodies, are released only upon hydrolysis of the first 1-3% of DNA. Further digestion with DNAase I causes no additional solubilization of these proteins. The chromatin fraction enriched in immunogenic proteins is also released upon autolytic digestion of chromatin with endogenous nuclease. The data obtained suggest that by their function tissue-specific and tissue-non-specific antigenic determinants belong to the same class of non-histone proteins localized in the chromatin sites hypersensitive to nucleases. The possible role of these proteins in regulation of the transcription is discussed.
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PMID:Immunochemical study of chromatin non-histone proteins. II. Localization of immunogenic tissue-specific proteins in nuclease-hypersensitive sites of chromatin. 674 31

Extracts of HeLa S3 cells were electrophoresed on polyacrylamide gels; gel slices were eluted and the eluates were assayed for DNase activities against native and denatured DNA substrates in the presence of MgCl2 or Na2EDTA. Aliquots of each eluate were also assayed for their ability to nick the circular supercoiled PM2 phage DNA to distinguish endonucleases from exonucleases. Peaks of endonuclease activities were characterized as forming 3'-phospho-oligonucleotides or 5'-phospho-oligonucleotides by the use of oligonucleotides produced by these enzymes as substrates for the 5'-phosphate-specific snake venom exonuclease. The total activity of DNases in gel eluates was much higher than that in cell extract applied to the gel, indicating the presence of inhibitors in the cell extract.
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PMID:Neutral deoxyribonucleases of HeLa S3 cells: electrophoretic separation, characterization, substrate specificity and mode of action. 677 64

Alkaline deoxyribonuclease activity was detected in culture of Mycoplasma pneumoniae. This enzyme required MgCl2 to stimulate its activity and preferred double-stranded to single-stranded DNA as substrate. By using polyacrylamide gel electrophoresis or isoelectric electrofocusing, two distinctive deoxyribonuclease activities were demonstrated both in cultured broth and cell homogenate.
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PMID:Detection of deoxyribonuclease activities of Mycoplasma pneumoniae. 679 Feb 44

Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease and ribonuclease, washing of residual nuclei with 0.5 M MgCl2, and discontinuous gradient centrifugation in buffered Ficoll solutions. Electron microscopic examination of the preparations showed single membrane and double membrane vesicles and membrane sheets. Pores or residual pores were often visible. In double membrane profiles the two unit membranes were often separated by the remains of the perinuclear cistern. The nuclear membrane fragments contained 58% protein, 23.8% phospholipid, 6% sterols, 7.1% neutral acylglycerols, 4.8% RNA, and 0.3% DNA. The phospholipid content of the membrane preparations was influenced by a phospholipase activity with acidic pH optimum.
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PMID:The isolation of Saccharomyces cerevisiae nuclear membranes with nuclease and high-salt treatment. 704 77

Two chromatin fractions were isolated from rat liver nuclei and from sperm of the sea urchin, Strongylocentrotus intermedius, by chromatin treatment with DNAase II with subsequent fractionation of the resultant fragments via their precipitation with MgCl2 solution. The analysis of distribution between these fractions of rapidly in vivo labeling RNA associated with chromatin and the in vitro investigation of template activity of isolated chromatin fractions allowed to conclude that one of the fractions is transcriptionally active and the other is inactive. The active chromatin fraction, similarly to the inactive one, contains all five histones. Unlike the latter, however, the former fraction is enriched in non-histone proteins and impoverished in histone H1. It is shown that the active chromation fraction of rat liver is composed of small fragments heterogenous in size. Rapidly synthesizing RNA in this fraction is mainly associated with mononucleosomal particles. Data on electrophoretic mobility of DNA fragments isolated from the active chromatin fraction from rat liver show that transcriptionally active sites of chromatin retain the nucleosomal structure.
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PMID:[Study of fractionated rat liver and sea urchin chromatin]. 718 19

Isolated nuclei incubated with [14C]protein hydrolysate are shown to incorporate labelled amino acids into the acid-insoluble fraction. Purified chromatin and the complex of DNA with firmly bound proteins possess similar ability. The optimum pH of the reaction is 6.5-7.0, 2 mM MgCl2 stimulates incorporation, the temperature optimum is 37-40 degrees C. Chloramphenicol depresses incorporation by 70%, puromycin by 40%, cycloheximide does not affect the chromatin activity. Incorporation does not depend on the presence of ATP or GTP, and is substantially inhibited by deoxyribonuclease but not by ribonuclease treatment of chromatin or of the nuclei. Specific activity of firmly bound chromatin non-histone proteins is higher than that of labile bound ones; histones are not labelled. After pronase treatment of proteins radioactivity changes to an acid-soluble state. The molecular weight of isolated labelled polypeptides is about 6000 as shown by gel filtration and the analysis of NH2-terminal amino acids. Labelled polypeptides firmly bound to DNA consist of 7-10 amino acids. Specific activity of proteins firmly bound to DNA increases linearly with the time of incubation of chromatin with [14C]protein hydrolysate, the activity curve of labile bound non-histone proteins has a distinct sygmoid character. The polypeptide-synthesizing activity of rat liver chromatin increases between 9 h and 21 h after partial hepatectomy. Irradiation with 800 rads 30 min before the operation prevents activation of amino acid incorporation. From nine amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated in the system described. Glutamic acid is polymerized most effectively. Glutamine, asparagine and glycine are incorporated 7-8 times less. The data are given indicating that the incorporation is not random when an amino acid mixture is present. Preincubation of chromatin with NAD+ but not with its analogues increases the polypeptide-synthesizing activity of chromatin. The activation is prevented by thymidine and nicotinamide. Storage (18 h at 2-4 degrees C) brings about a complete loss of the polypeptide-synthesizing activity of chromatin. The ability of 'old' chromatin to incorporate amino acids can be restored by preincubating it with NAD+. Storage of chromatin in the presence of 5 mM adenosine 3',5'-monophosphate (cAMP) does not result in decrease of the polypeptide-synthesizing activity. It is assumed that poly-(ADP-ribose) is the energy source for amino acid activation in the system described.
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PMID:Polypeptide-synthesizing activity of eukaryotic chromatin. Properties, dependence on poly(ADP-ribose) and connection with the cell cycle. 737 37

Transcriptionally active and inactive chromatin fractions were isolated from Physarum polycephalum after depolymerization of chromatin with DNAase II or micrococcal nuclease, followed by fractionation in 5 mM-MgCl2. The active fraction of chromatin comprised up to 21% of nuclear DNA and was enriched 22-fold in the labelled nascent RNA. Both chromatin fractions were shown to have the nucleosomal structure. DNA of the active fraction of chromatin was degraded much faster with DNAase I and micrococcal nuclease than the DNA of the inactive fraction.
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PMID:Isolation and susceptibility to nucleases of transcriptionally active and inactive chromatin fractions from Physarum polycephalum. 743 80


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