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:3.1.22.1 (DNase II)
429 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg(2+)-dependent and DNase II-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast, a clear difference was shown in the circulating CD34+ cells, in that only Mg(2+)-dependent, Ca(2+)-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg(2+)-dependent and acidic endonuclease concomitantly with the disappearance of the Mg(2+)-dependent endonuclease when CD34+ cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg(2+)-dependent nuclease activity. Our results suggest an association of the Mg(2+)-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation.
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PMID:Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different between human CD34+ cells and their granulocytic descendants. 754 3

Activation of a triplet of nuclear proteins (NP42-50) was observed in human Jurkat T cell line following treatment with an antibody to CD95 (Fas/Apo-1), a cell surface molecule involved in apoptotic cell death. The nuclease activity, corresponding to a triplet of proteins observed at approximately 42, 45, and 50 kDa in size, was extractable, heat-stable, and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing deoxyribonucleic acids (SDS-PAGE-DNA) assay. The NP42-50 activity requires the presence of Mg2+/Ca2+ and is insensitive to inactivation by heating at 80 degrees C for 5 min. Zinc effectively inhibited the enzymatic activity of NP42-50 on SDS-PAGE-DNA and also protected Jurkat cells from the CD95-mediated apoptosis in cell cultures. The nuclease activation, however, was not a unique pathway for the CD95-mediated cell death. The apoptosis induced by arabinofuranosyl cytosine, a chemotherapeutic agent, also activated the NP42-50 nuclease activity in Jurkat cells, suggesting that a similar cascade of subsequent events in apoptosis may occur in most instances although many different signals can initiate apoptotic cell death in various cell types. The nuclease identified by this study appears to be distinguishable from DNase I or DNase II by its molecular characteristics and its enzymatic requirements. The NP42-50, with respect to the nuclease activity closely associated with apoptotic cell death, may serve as a candidate for the endonuclease(s) involved in the cleavage of DNA into fragments during apoptosis.
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PMID:A triplet of nuclease proteins (NP42-50) is activated in human Jurkat cells undergoing apoptosis. 755 79

Apoptosis is a pathway of cell death characterized by internucleosomal digestion of genomic DNA. Such DNA digestion can be induced by both physiological stimuli and cytotoxic treatment with many anticancer agents. This digestion has generally been considered to be mediated by a Ca2+/Mg(2+)-dependent endonuclease that is activated by increases in intracellular Ca2+. However, we suggest that an alternate endonuclease, DNase II, may be a more likely candidate. In these studies, apoptosis was induced in human HL-60 cells by a 30-min incubation with the topoisomerase II inhibitor etoposide. DNA digestion characteristic of apoptosis began within 3 h of removal of etoposide. Morphological indication of apoptosis was observed concurrently. Only about 20% of the cells underwent apoptosis at this time; these appeared to be cells in S phase at the time of etoposide treatment. The remainder of the cells progressed to the G2 phase and arrested there for at least 48 h. Intracellular Ca2+ and pH were measured in individual cells by flow cytometry. No changes in intracellular Ca2+ were observed, but an acidification of up to 1 pH unit occurred in about 15% of the cells and correlated with the time course of appearance of DNA digestion. Cells were sorted on the basis of intracellular pH and only the acidic cells showed the morphology and DNA digestion characteristic of apoptosis. These results demonstrate the involvement of DNase II in apoptotic DNA digestion and suggest mechanisms of pH homeostasis as regulators of apoptosis.
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PMID:Etoposide-induced apoptosis in human HL-60 cells is associated with intracellular acidification. 838 92

Cell death occurs by apoptosis during programmed deletion of cells and following exposure to cytotoxic agents. Central to the mechanism of apoptosis is internucleosomal DNA digestion by an endogenous endonuclease which is thought to mediate cell death. An axiom of apoptosis is that the endonuclease involved is a Ca2+/Mg(2+)-dependent endonuclease. During purification of endonucleases from Chinese hamster ovary cells, we found little Ca2+/Mg(2+)-dependent endonuclease activity, but large amounts of an endonuclease active below pH 7. This acidic endonuclease was activated in intact cells by reducing intracellular pH values below 7 with a proton ionophore. This activity generated internucleosomal digestion of DNA characteristic of apoptosis. Nuclear extracts contained a cation-independent endonuclease with identical pH-dependent activity. We have compared the acidic endonuclease to bovine deoxyribonuclease II (DNase II) and have found them nearly identical by all tests, including sensitivity to various inhibitors, purification by the same chromatographic steps, and recognition by antibody raised against the bovine enzyme. Addition of either the acidic endonuclease or bovine DNase II to isolated nuclei induced internucleosomal DNA digestion up through pH 6.5. These data demonstrate that DNase II can mediate internucleosomal DNA digestion characteristic of apoptosis following intracellular acidification. Furthermore, these data question the premise that the Ca2+/Mg(2+)-dependent endonuclease is the only endonuclease involved in apoptosis.
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PMID:Identification of deoxyribonuclease II as an endonuclease involved in apoptosis. 842 78

The nature of the endonucleases responsible for DNA fragmentation in apoptosis has not yet been clearly defined. The intracellular acidity has been known to greatly affect apoptosis probably by affecting the activity of the endonucleases. In this study, the implication of pH in the apoptosis was investigated through the use of human HL-60 leukemia cells. The cells were incubated in media with different pH ranging from 3.5 to 7.5 for 4 hrs and the mode of cell death was investigated. The trypan blue exclusion assay showed that close to 25% and 90% of the cells were dead when incubated in pH 6.4 and pH 5.0 media, respectively. The agarose gel electrophoresis of DNA demonstrated that significant DNA fragmentation occurred in the HL-60 cells incubated in the pH 6.2-6.4 media for 4 hr indicating cell death by apoptosis. The electron microscopy study also demonstrated that many of the cells incubated in the pH 6.4 medium were in the process of apoptosis while the cells maintained in the pH 5.0 medium were dying by necrosis. The intracellular pH (pHi) of HL-60 cells was 6.6-6.9 when the extracellular pH (pHe) was 6.2-6.4. These results demonstrated that DNase I which has a maximal endonuclease activity near pH 7.0 may be responsible for the apoptosis accompanied by DNA fragmentation in HL-60 cells in the pH 6.4 medium. This observation is at variance with the previous reports that DNase II mediate the DNA fragmentation in apoptosis. The cell death at extremely low pH (pH 5.0) appeared to be due mainly to necrosis.
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PMID:Effects of intracellular pH on apoptosis in HL-60 human leukemia cells. 859 48

DNA fragmentation is a common biochemical hallmark of apoptosis. It is catalyzed by endogenous Ca2+, Mg(2+)-dependent endonuclease(s). Although the exact identity of the apoptotic endonuclease is still a matter of debate, a number of candidate nucleases have been proposed like NUC18, DNase II and DNase I. Relatively large amounts of nucleases are also expressed by mycoplasmas, cell wall-less bacteria of the class Mollicutes, which are found as contaminants in up to 45% of the continuous cell lines in current use. In order to clarify the effect of these pathogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expression) and morphological changes characteristic of apoptosis (cell shrinkage, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis-free and -infected cultures of the human pancreatic adenocarcinoma cell line PaTu 8902 and of mouse NIH 3T3 fibroblasts. For that purpose we employed cells cultured under standard conditions and cells exposed to the protein synthesis inhibitor cycloheximide, which is known to induce apoptosis in various cell systems. After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA degradation. In contrast, nuclease activities in the molecular range of 47 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative. The expression of the nucleases and the cycloheximide-induced DNA fragmentation were suppressed by the prokaryote-specific protein synthesis inhibitor chloramphenicol. Moreover, partially purified nucleases from supernatants of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites. These data indicate that DNA ladder formation in cell culture systems can also be caused by mycoplasmal nucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress. Therefore, internucleosomal DNA fragmentation in established cell lines has to be regarded with care, unless mycoplasmal infection can be excluded, or the existence of endogenous endonucleases can be proven. The presence of endonucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M. hyorhinis. In contrast, the expression of an apoptotic morphology was not restricted to infected cells; in both mycoplasma-free and -contaminated cultures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342. Electron microscopic studies revealed that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells. Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis as indicated by the parallel occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).
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PMID:Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases. 888 84

Xib, a gene recently reported to reside on the q28 region of the human X chromosome [Pergolizzi et al. (1996) Gene 168, 267-270], contains an open reading frame homologous to those of the DNase I family enzymes. The full open reading frame of this gene has been fused to the E. coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein. The partially purified chimeric protein is enzymatically active. It introduces single and double stranded breaks into supercoiled DNA, at 30 degrees C in the absence of divalent cations and at a pH optimum of 5.2. To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.
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PMID:Functional characterization of a human DNase-like protein encoded by a gene positioned in Xq28. 909 69

A polyclonal antibody against purified bull seminal plasma Ca2+, Mg2+-dependent endonuclease was raised in a rabbit. The antibody specifically cross-reacted with chromatin-bound Ca2+, Mg2+-dependent endonucleases from bovine thymus, human placenta, and bovine, rat and mouse liver in addition to the bovine seminal enzyme. The antibody did not cross-react with other endonucleases examined, including the acid-endonucleases from bovine thymus and liver, porcine spleen DNase II, micrococcal nuclease, and bovine pancreas DNase I, a known Ca2+ and Mg2+ requiring endonuclease. The present results indicate that this antibody specifically recognizes a class of so-called Ca2+, Mg2+-dependent endonuclease, which is localized in cell nuclei of various tissues and is probably involved in chromatin degradation during apoptosis. The antibody will be used to study the functional role of this class of endonuclease.
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PMID:Anti-Ca2+, Mg2+-dependent endonuclease antibody detects specifically a class of chromatin-bound endonuclease. 924 Apr 53

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.
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PMID:On the use of Zn2+ to discriminate endonucleases activated during apoptosis. 935 93

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cation-independent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.
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PMID:L-DNase II, a molecule that links proteases and endonucleases in apoptosis, derives from the ubiquitous serpin leukocyte elastase inhibitor. 958 2


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