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1.
Spectra of thymocyte's nuclear nucleases of control and glucocorticoid treated (5 mg/kg body weight) adrenalectomized rats have been investigated. Using the method of SDS-electrophoresis of nuclear proteins in 3H-DNA-polyacrylamide gel (PAAG) the authors managed to discover a number of polypeptides of 35, 32, 17.7, 17.0, 16.4 kDa molecular mass possessing a nuclease activity. The enzyme of 35 kDa is only active in the presence of Ca2+ and Mg2+ ions and inhibited by cycloheximide. Nucleases of 32, 17.7, 17.0, 16.4 kDa are active in the presence of Ca2+ ions. The enzymic activity of these nucleases increases 60 min after steroid treatment. Nuclease of 17.7, 17.0, 16.4 kDa are poly(ADP-ribosylated). Glucocorticoid mediated activation don't blocked by poly(ADP-ribosylation). Possible role and mechanism of discovered nucleases are discussed. 相似文献
2.
Spectra of thymocyte nuclear DNAases of control and irradiated (4 Gy) rats have been investigated. Using the method of SDS-electrophoresis of nuclear proteins in DNA-polyacrylamide gel (PAAG) the authors managed to discover a number of polypeptides of 35, 32, 17.7, 17.2, and 16.4 kDA molecular mass possessing a DNAase activity. The enzyme of 35 kDA is only active in the presence of Ca2+ and Mg2+ ions. Nucleases of 32, 17.7, 17.2, and 16.4 kDA are active in the presence of Ca2+ ions and inactive in the presence of Mg2+ ions or in the absence of divalent cations. A simultaneous addition of Ca2+ and Mg2+ ions to the incubation medium causes a synergistic effect with respect to the manifestation of these DNAase activities. Nucleases of 32, 17.7, 17.2, and 16.4 kDa only emerge after the preliminary removal of histones by ion exchange chromatography on a column with CM-sephadex C-50. The enzymic activity of 32 kDA protein increases 60 min after irradiation and drops to the control value in 4 h. At the same time, the postirradiation increase in DNAase activity of a low-molecular weight enzyme group remains invariable throughout the entire period of observation (1-4 h). The preinjection of cycloheximide (CHI) prevents the postirradiation degradation of chromatin and, simultaneously, makes the enzymic activity, corresponding to 35 kDA protein, disappear at the electrophoregrams. The experiments with CHI permit to identify the given enzymic fraction as Ca/Mg-dependent endonuclease. This indicates the participation of normally pre-existing Ca/Mg-dependent endonuclease in implementing the process of chromatin enzymic degradation in the irradiated thymocytes. 相似文献
3.
L.V. Nikonova P.A. Nelipovich S.R. Umansky 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(3):281-289
Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes — a Ca2+/Mg2+-dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca2+/Mg2+-dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca2+/Mg2+-dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg2+-dependent nucleases was shown to increase (by 40 and 50%, respectively) 3 h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca2+/Mg2+-dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes. 相似文献
4.
V. M. Ermekova O. S. Melkonyan R. N. Zotova L. F. Nararova S. R. Umansky 《Molecular biology reports》1984,9(4):263-267
Localization of immunogenic tissue-specific proteins in chromatin regions, hypersensitive to endogenous nucleases, has been studied using rabbit antibodies against rat thymus chromatin. It is shown that the first 1–2,5% of the chromatin (calculating on DNA), released by Mg2+-, Mn2+-, and Ca2+/Mg2+-dependent nuclear endonucleases are drastically enriched in tissue-specific antigenic determinants. The released chromatin fractions are found to contain a heterogeneous set of nonhistone proteins and are deficient in histones. The cleavage of nuclear DNA by endogenous acidic nuclease, independent on bivalent ions, resulted in a significantly less enrichment of the released fractions with immunogenic proteins. 相似文献
5.
Besides the well-known heat-stable extracellular staphylococcal nuclease (EC 3.1.4.7) and cell surface bound nuclease, one more nuclease, which is heat-labile, has been identified and purified on phosphorylated cellulose column and characterized. Analyses by Sephadex G-75 gel chromatography indicates that the heat-labile cellular nuclease has molecular weight of about 16,000 similar to those of extracellular and cell-surface bound nucleases. Like the heat-stable nucleases, the heat-labile enzyme acts on both DNA and RNA, is more active on heat-denatured DNA, requires Ca2+ ions for activity and maximum catalytic activity is observed at pH 9.8–10 and at 45°C. The results suggest that the three enzymes have properties strikingly similar to one another and therefore may be related structurally. 相似文献
6.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2019,1866(7):1180-1188
Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca2+ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a ‘clickable’ ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N3-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca2+ responses. Higher concentrations of 5N3-8-ethynyl were required to elicit Ca2+ release or displace 32P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of 32P-5N3-8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional 32P-5N3-NAADP, the clickable 32P-5N3-8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the ‘clickable’ ethynyl moiety. No proteins were specifically labeled by 32P-5N3-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N3-8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a ‘bifunctional’ probe with utility for isolating NAADP-binding proteins. 相似文献
7.
Haiyan Zhao Zihan Lin Anna Y. Lynn Brittany Varnado John A. Beutler Ryan P. Murelli Stuart?F.?J. Le?Grice Liang Tang 《Nucleic acids research》2015,43(22):11003-11016
Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. 相似文献
8.
The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca2+, Mg2+ and Mn2+; only the nucleases active at neutral pH were inhibited by Zn2+. The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture. 相似文献
9.
Carmen Perez-Terzic Marisa Jaconi David E. Clapham 《BioEssays : news and reviews in molecular, cellular and developmental biology》1997,19(9):787-792
In eukaryotic cells the nucleus and its contents are separated from the cytoplasm by the nuclear envelope. Macromolecules, as well as smaller molecules and ions, can cross the nuclear envelope through the nuclear pore complex. Molecules greater than approx. 60 kDa and containing a nuclear localization signal are actively transported across the nuclear membranes, but there has been little evidence for regulatory mechanisms for smaller molecules and ions. Recently, diffusion across the nuclear envelope has been observed to be regulated by nuclear cisternal Ca2+ concentrations. Following depletion of Ca2+ from the nuclear store by inositol 1,4,5-trisphosphate or Ca2+ chelators, a fluorescent 10 kDa marker molecule was no longer able to enter the nucleus. Distinct conformational states of the nuclear pore complexes depended on the Ca2+ filling state of the nuclear envelope, supporting the assumption that a switch in the conformation of the nuclear pore complex may control the transport of intermediate-sized molecules across the nuclear envelope. Thus nuclear Ca2+ stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. The physiological significance of this finding is currently unknown. 相似文献
10.
M. Vischi S. Marchetti 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,95(1-2):185-190
Electroporation is becoming an increasingly important technique for plant transformation. Nevertheless, no positive results
were achieved in barley when uninucleate microspores were used as target cells. Since it was previously demonstrated that
electric shocks create pores in the microspore cell wall, experiments were designed to verify the presence of nucleases in
the electroporation mix. Aliquots of all the solutions used for microspore extraction, purification and transformation were
collected and analysed using supercoiled pBI 221 as a substrate; a nuclease activity was detected in all samples. Though microspore
rinsing removed most nucleolytic activity in the supernatants, DNA preservation in the electroporation buffer was difficult
to achieve, because microspores appeared capable of synthesising and releasing endonucleases at any time. Microspore chilling
at 0°C was fairly effective in reducing nuclease secretion in the mix, whereas 1%PEG or 10 mM EDTA maintained most of the
DNA in a supercoiled or circular relaxed form. EDTA effects were counterbalanced by Mg2+, but not Ca2+ or Zn2+, and enhanced by Mn2+. Barley microspore nucleases actively degraded different DNAs as well as TMV RNA, and apparently had a molecular weight above
30 kDa. Nuclease inactivation with EDTA did not alter microspore viability and allowed a transient expression of the uidA gene in electroporated barley microspores.
Received: 13 January 1997 / Accepted: 28 February 1997 相似文献
11.
12.
Androgen treatment protects mouse liver chromatin from cleavage by endogenous nucleases during aging
We have examined the endogenous nuclease activity of the liver of intact, castrated and testosterone-treated mice of different ages. Both Mg2+- and Ca2+-dependent endogenous nuclease activities decline in old age. Withdrawal of the hormone increases nuclease activity in the immature and young. However, testosterone administration prevents the digestion of nuclei to different extents in all ages. These findings suggest a possible protective role of testosterone in the cleavage of liver chromatin by endogenous nucleases during the aging of mice. 相似文献
13.
Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were
about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions
were as active to UV-irradiated (270 J m-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high
concentrations of EDTA, NaCl, Mn2+, Ca2+, Zn2+ ions and by N-ethylmaleimide. They do not require Mg2+ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide
pH range (5.5–9), the optimum for DNase action in the presence of Mg2+ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely
that DNase acts in the endonucleolytic way. 相似文献
14.
A manganese-stimulated exonuclease was purified from culture fluids ofBacillus subtilis 168 using ammonium sulfate fractionation, SephadexG-150 gel filtration, and DEAE-Sephadex ion-exchange chromatography. This extracellular nuclease was found to attain maximal activity in the presence of 5 mM Mn2+. Little or no activity was demonstrated in the presence of Fe2+, Mg2+, Zn2+, Cu2+, or Ni2+, but the nuclease was somewhat active with Ca2+. The nuclease exhibited a broad pH range, with maximum activity at pH 8.5. A molecular weight of 214,000 was calculated for the protein using Sephacryl S-300 columm chromatography. Incubation of the enzyme with the closed circular DNA of plasmid pUB110 indicated that the nuclease is strictly, exonucleolytic in nature. 相似文献
15.
《Cell calcium》2019
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP. 相似文献
16.
Tritiated analogues of the Ca2+ channel blockers such as [3H] PN200-110, [3H] verapamil and [3H] diltiazem have been used to identify and isolate Ca2+ antagonist receptors. The Ca2+ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca2+ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction. 相似文献
17.
Surface-Bound Nuclease of Staphylococcus aureus: Purification and Properties of the Enzyme
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The surface-bound nuclease of Staphylococcus aureus liberated during formation of protoplasts was purified 1,000-fold by chromatography on phosphocellulose. Its properties were compared with those of the known extracellular nuclease, purified 200-fold by the same procedures. The adsorbance of the surface-bound nuclease on phosphocellulose was distinctly different from that of the extracellular nuclease, but other properties of the two enzymes were similar. Both enzymes had a pH optimum of about 10 and required Ca2+ for activity. Both enzymes hydrolyzed deoxyribonucleic acid (DNA) and ribonucleic acid, and denatured DNA was a better substrate than native DNA. Both enzymes were inhibited by the same metal ions. Nuclease-less mutants of S. aureus were isolated from S. aureus 209P by using N-methyl-N′-nitroso-N-nitrosoguanidine. These mutants contained neither surface-bound nor extracellular nuclease activity. These results suggest that the surface-bound and extracellular nucleases are expressed from the same cistron of S. aureus. 相似文献
18.
I. A. Dvortsov N. A. Lunina V. V. Zverlov G. A. Velikodvorskaya 《Molecular Biology》2010,44(4):591-595
Endo-β-1.3–1.4-glucanase Lic16A of the moderate thermophilic anaerobe Clostridium thermocellum has a complex multimodular structure. In addition to the catalytic module, Lic16A contains eight auxiliary modules, five
of which are substrate-binding modules. The new family 54 substrate-binding module CBM54 (25.2 kDa), which is at the N terminus
of the enzyme, has a cleavage site in its N-terminal part, and its cleavage yields a shortened module CBM54C (17.2 kDa) in
vivo and in vitro. CBM54C was cloned in Escherichia coli and purified to electrophoretic homogeneity. The binding constants of CBM54C to xylan, chitin, insoluble yeast cell wall
β-glucan, and bacterial crystalline cellulose were of the same order of magnitude as for CBM54. However, CBM54C did not bind
pustulan, avicel, and chitosan, in contrast to CBM54. Calcium ions restored the ability of CBM54C to bind these three carbohydrates.
CBM54 substrate binding promiscuity suggested multiple binding sites, some of them being Ca2+ dependent. The Ca2+-independent sites for avicel, pustulan and chitosan were localized to the spontaneously split-off N-terminal part (8 kDa)
of CBM54. The arrangement of Ca2+-dependent and Ca2+-independent binding sites for various substrates was suggested. 相似文献
19.
Ki-Young Lee Kyu-Yeon Lee Ji-Hun Kim In-Gyun Lee Sung-Hee Lee Dae-Won Sim Hyung-Sik Won Bong-Jin Lee 《Nucleic acids research》2015,43(10):5194-5207
HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn2+ and Mg2+ ions, respectively, and decreased by Cu2+ ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. 相似文献
20.
Irina A. Kostrikina Marina E. Kolesova Irina A. Orlovskaya Valentina N. Buneva Georgy A. Nevinsky 《Journal of molecular recognition : JMR》2011,24(4):557-569
It has been shown for the first time that polyclonal IgG abzymes (Abzs) with DNase activity from the sera of autoimmune‐prone MRL/MpJ‐lpr mice can be separated by isoelectric focusing into many subfractions having the isoelectric points (pI) from 4.5 to 9, with the maximal activity for Abzs with pI = 6.5–9.0. Affinity chromatography on DNA‐cellulose separated DNase IgGs into many subfractions demonstrating a range of affinities for DNA and different levels of the relative DNase activities (RDA) due to intrinsically bound metals and after addition of external Mg2+, Mn2+, Ca2+, and Mg2++Ca2+. Some fractions significantly increase RDAs in the presence of external ions (Mg2++Ca2+ > Mg2+ > Mn2+ > Ca2+), while each of this cofactor can also inhibit or have no influence on the RDAs of another fractions. It is known that complexes of DNA with histones and other proteins of apoptotic cells are the primary immunogens in systemic lupus erythematosus (SLE). Bovine serum albumin (BSA) and methylated BSA (mBSA) increase the RDAs of only some fractions, while have no effect or inhibit other IgG fractions. The ratio of the RDAs in the presence of all metal ions, BSA, and mBSA was individual for every abzyme fraction. Mn2+ and Ca2+ stimulated accumulation of only relaxed form of supercoiled DNA (scDNA) in the case of all subfractions, while in the presence of Mg2+ antibodies (Abs) of some subfractions (and in the presence of Mn2+ +Ca2+ all subfractions) produced relaxed DNA (rDNA) and linear DNA (linDNA) in a variable extent. The data obtained show that the polyclonal Abzs of mice may be a cocktail of Abs directly to DNA, RNA, and their complexes with proteins and anti‐idiotypic Abs to active centers of different nucleases. The diversity of the physicochemical and kinetic characteristics of the Abzs seems to be significantly widened when pre‐diseased mice spontaneously develop the disease. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献