Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.
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PMID:Poly(adenosine dephosphate ribose) metabolism and regulation of myocardial cell growth by oxygen. 2 65

Isolated nuclei from HeLa cells can incorporate labeled ADP-ribose from NAD into an acid-precipitable product, poly(ADP-ribose). This reaction is stimulated by 4-6-fold by the addition of deoxyribonuclease I to the complete reaction mixture. If the nuclei are treated first with deoxyribonuclease I, no effect is seen; the stimulation is only apparent when the two enzymes deoxyribonuclease I and poly(ADP-ribose) polymerase, are operating at the same time. After making several minor modifications in the assay mixture, it was found that another endonuclease, micrococcal nuclease, can also stimulate the poly(ADP-ribose) polymerase activity of HeLa nuclei. A comparison of the two stimulatory effects indicated that the two endonucleases activated to the poly(ADP-ribose) polymerase activity of HeLa nuclei in the same way. Overall this evidence suggests that poly(ADP-ribose) polymerase may have a functional role in the process of DNA repair.
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PMID:Stimulation of nuclear poly (adenosine diphosphate-ribose) polymerase activity from HeLa cells by endonucleases. 16 97

The distribution of a chromatin-bound, nuclear protein modifying enzyme, poly (adenosine diphosphate-ribose) polymerase, and its product, poly(ADP-ribose), among various fractions of sheared and nuclease-digested HeLa cell chromatin has been examined. Epichlorohydrin-tris(hydroxymethyl)aminomethane-cellulose and glycerol gradient fractionation of solubilized chromatin indicated that poly(ADP-ribose)polymerase activity was associated primarily with the template active regions (euchromatin), whereas the transcriptionally inert chromatin fractions were found to contain relatively low levels of ADP-ribosylating activity. When isolated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers (v bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the 11S monomers was found to contain appreciable poly(ADP-ribose) polymerase activity. If, on the other hand, isolated HeLa cell nuclei were first incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, it was found that those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and that a considerable portion of the homopolymeric product is ultimately associated with the 11S v bodies. Additional evidence is presented which indicates that the absence of nucleosome monomer-associated poly(ADP-ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures, and that the activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves.
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PMID:Poly(adenosine diphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure. 18 3

We present evidence that T3 can alter the ADP-ribosylation of chromatin associated proteins. Nuclei from GH1 cells were incubated with [adenylate-32P]NAD and the radioactivity incorporated into histone and non-histone proteins was quantitated and analyzed by gel electrophoresis and autoradiography. Incubation of GH1 cells for 24 h with T3 lowered by 40-70% the [32P]ADP-ribose incorporated into nuclear proteins. However, incubation for 3 h with T3 resulted in a stimulation instead of a decrease of in vitro [32P]ADP-ribose incorporation. The major ADP-ribosylated component electrophoresed as a 120,000 molecular mass non-histone protein, and radiolabeled histones were also observed. The same protein species were observed for all the experimental groups and T3 affected the extent of ADP-ribosylation but did not alter the sedimentation of the [32P]ADP-ribosylated components excised from chromatin after micrococcal nuclease digestion.
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PMID:Influence of thyroid hormone on ADP-ribosylation of nuclear proteins in cultured GH1 cells. 200 28

Incubation of GH1 cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP.
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PMID:Cholera toxin affects nuclear ADP-ribosylation in GH1 cells. 282 73

HeLa cell nuclei with DNA labeled with [3H] thymidine have been preincubated under varying conditions and then incubated with micrococcal nuclease. Aliquots, removed at increasing times, were analyzed for mononucleosomal size DNA and for acid-soluble DNA, the ratios were plotted and a slope was determined. Preincubation with ATP and a regenerating system increased the slope 2 fold. Optimum ATP concentrations were above 0.25 mM. The ATP effect was reversed by novobiocin. No inhibition of the ATP effect was observed with nalidixic acid, coumermycin, oxolinic acid, VM-26, aphidicolin, or 3 amino-benzamide. NAD or cAMP or cGMP had no effect with or without ATP. Other nucleoside triphosphates could substitute to varying degrees for ATP as could ATP analogues. Nuclei from log phase cells showed no ATP effect, but log phase cells, partially depleted of ATP by incubation with deoxyglucose, showed the effect. The effect was lost in nuclei on long-term storage. No evidence was found for differential degradation of core histones, histone H-1 or DNA, and there was no evidence of nucleosome sliding.
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PMID:The effect of preincubation of HeLa cell nuclei with ATP on the degradation of mononucleosomal DNA by micrococcal nuclease. 301 48

The nuclear location of NMN adenylytransferase, which catalyses the formation of NAD and pyrophosphate from ATP and NMN, has been examined to ascertain if the enzyme is bound to the domains of chromatin which undergo poly(ADP-ribos)ylation. This latter reaction utilizes much of the cellular NAD. A radioisotope assay using [alpha-32P]ATP was developed to enable precise measurement of picomole amounts of NAD. With this assay, it appeared that the reaction catalysed by NMN adenylyltransferase proceeded with a rapid, early 'burst' of NAD before steady-state velocities were established. From this it was calculated that there could be 10- active sites of NMN adenylyltransferase per HeLa nucleus in asynchronously growing cells: that is, approximately one per 10-20 nucleosomes. Very little enzyme activity was liberated by digesting HeLa nuclei with micrococcal nuclease in 80 mM NaCl, and the enzyme which was solubilized was not bound to oligonucleosomes separated by electrophoresis on polyacrylamide gels. In contrast, poly(ADP-ribose) polymerase activity was clearly demonstrated on these particles. The enzyme was readily liberated by DNase I digestion, especially when the digestion was carried out in low-ionic-strength buffer. The results demonstrated that the enzyme was neither bound to oligonucleosomes nor part of the nuclear envelope or matrix. Preliminary results suggested that there could be some direct channelling of NAD between the two enzymes in intact nuclei. It appears that NMN adenylyltransferase is bound within rather than to chromatin.
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PMID:NMN adenylyltransferase: its association with chromatin and with poly(ADP-ribose) polymerase. 629 57

It has not previously been possible to label the nuclear protein modification poly(ADP-ribose) directly from NAD because of the impermeability of the cell membrane. We have overcome this important problem by micro-injection of radioactively labelled NAD into Xenopus laevis early embryos. The polymer was identified and then quantified by its insensitivity to DNAase, RNAase, and spleen phosphodiesterase and by the chromatographic mobility of the products of digestion with snake-venom phosphodiesterase. The quantity of poly(ADP-ribose) present after 25 h of development (129 ng/mg DNA) is lower than that found in fully differentiated tissue.
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PMID:Direct radioactive labelling of poly(ADP-ribose) in developing Xenopus laevis embryos. 630 67

Poly(ADP-ribose) [poly(ADP-Rib)] polymerase of HeLa nucleosomes has been shown in vitro, to catalyze the synthesis of a complex of histone H1 containing 2 H1 histones and 15-16 units of oligo(ADP-Rib). The synthesis of the H1 complex in vitro was compared in polynucleosome populations of various sizes (3--16 and greater than 30) released from HeLa nuclei following micrococcal nuclease digestion. Poly(ADP-Rib) was synthesized from [32P]NAD and the poly(ADP-ribosyl)ation of H1 was studied by selective H1 extraction, gel electrophoresis and autoradiography. Quantitative differences in H1 complex formation occurred when either chromatin concentration or polynucleosome length was varied. The data indicated that H1 complex formation in vitro was favored in polynucleosomes 16 nucleosomes long as compared to 8 nucleosomes. A series of partially ADP-ribosylated H1 species was also detected. Partially modified H1 species migrate more slowly than pure H1 in dodecylsulfate gels. The reduced mobility is a function of the number of attached ADP-Rib moieties. Thus, molecules containing one molecule of H1 and various numbers of ADP-Rib residues can be separated. When the partially modified H1 species were incubated in alkali to cleave the linkage of ADP-Rib to protein, (ADP-Rib1-15) were detected by chain length analysis on 15% polyacrylamide gels. The intermediate H1 species could be chased, in vitro, into as H1 complex with NAD and thus were determined to be successive precursors in the formation of the H1 complex. Evidence is presented that the H1 complex is synthesized in intact cells permeabilized with lysolecithin.
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PMID:Characterization of poly(ADP-ribose)--histone H1 complex formation in purified polynucleosomes and chromatin. 746 Sep 42

Poly(ADP-ribose) polymerase (PADPRP) catalyzes the transfer of multiple ADP-ribose units from NAD to nuclear histone and nonhistone proteins, a reaction that appears to be important in the rejoining of DNA strand breaks during DNA repair and replication. We previously established and characterized a HeLa cell line that was stably transfected with a recombinant expression plasmid containing the mouse mammary tumor virus promoter upstream of a construct encoding PADPRP antisense RNA. We now show that after depletion of PADPRP mRNA as a result of antisense RNA expression, normal PADPRP mRNA concentrations are restored between 8 and 16 h after removal of dexamethasone (which activates the mouse mammary tumor virus promoter). By depleting antisense cells of PADPRP, we demonstrated the contribution of this enzyme to various aspects of nuclear structure and function: (a) amplification of a selectable gene encoding three early enzymes in the pyrimidine biosynthetic pathway was greatly increased in cells depleted of PADPRP; (b) chromatin structure was significantly altered in PADPRP-depleted cells, as indicated by reduced initiation and elongation of poly(ADP-ribose) chains attached to various nuclear protein acceptors, lower levels of poly(ADP-ribosyl)ation of histone H1, and an increased susceptibility of DNA to micrococcal nuclease digestion; and (c) the survival of PADPRP-depleted antisense cells exposed to the DNA alkylating and carcinogenic agent methyl methanesulfonate or nitrogen mustard was significantly reduced relative to that of control cells.
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PMID:Depletion of nuclear poly(ADP-ribose) polymerase by antisense RNA expression: influences on genomic stability, chromatin organization, and carcinogen cytotoxicity. 806 55


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