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:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An important role in the control of gene expression has been attributed to phosphoproteins present among chromatin non-histone proteins. In a previous work we have shown that at least part of these phosphoproteins are associated with nucleosomes. In this work we wanted to establish whether this association occurs with all nucleosomes or with the nucleosomes present in fragments preferentially released by a mild micrococcal nuclease digestion, which originated essentially from active parts of chromatin. Phosphoproteins were labelled in vivo by incubating hepatoma tissue-cultured cells with [32P]phosphate and chromatin was submitted to a limited micrococcal nuclease digestion. The released fragments were fractionated by preparative gel electrophoresis. [32P]Phosphoproteins were essentialy found in the smallest released fragments: monomers and dimers of nucleosomes. The same result was obtained when the phosphoproteins were labelled in vitro by incubating each fragment obtained by the preparative electrophoresis in the presence of [gamma-32P]ATP. It indicates that part of the protein kinase activity was strongly bound to the particles. The bound phosphoproteins were analysed by sodium dodecylsulfate/polyacrylamide gel electrophoresis. Two main polypeptides were characterized: phosphopeptide a, Mr 41000, present in all small fragments; phosphopeptide b, Mr 31000, present in all small fragments, except in the fastest moving nucleosomes. Phosvitin kinase was found associated with the small released fragments, its specific activity was by far the highest in the fraction which includes the dimers of nucleosomes. It is concluded that phosphoproteins and protein kinases are associated with the nucleosomes of the active parts of chromatin, which suggests a role of these proteins in the control of gene expression.
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PMID:Localization of phosphoproteins and of protein kinases in chromatin from hepatoma tissue-cultured cells. 743 87

The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA. 803 23

The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV circular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure.
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PMID:Fluorescence dynamics of staphylococcal nuclease in aqueous solution and reversed micelles. 814 63

The 32P-postlabelling methodology for analysis of DNA adducts derived from carcinogens containing one aromatic ring (e.g., safrole, styrene oxide, benzene metabolites, 1-nitrosoindole-3-acetonitrile) or a bulky non-aromatic moiety (e.g., mitomycin C, diaziquone) is reviewed. Six steps are involved: digestion of DNA to 3'-nucleotides, enrichment of adducts, 32P-labelling of adducts, separation of labelled adducts by TLC, detection, and quantitation. The first step, DNA digestion with micrococcal nuclease and spleen phosphodiesterase, is applicable to DNA modified with most carcinogens independent of their size and structure. Of the two commonly used procedures for enrichment of aromatic adducts in DNA digests, the nuclease P1 treatment is substantially more effective than butanol extraction for small aromatic and bulky non-aromatic adducts. For initial purification of these adducts from unadducted material after 32P-labelling, multi-directional polyethyleneimine (PEI)-cellulose TLC using 1 M sodium phosphate, pH 6.0, as the D1 solvent is not suitable, because they are not retained on PEI-cellulose under these conditions. A higher concentration of sodium phosphate (e.g., 2.3 M) or development with D1 and D3 solvents in the same direction helps to retain adducts of safrole and of benzene metabolites. Also, transfer of adducts from multiple cut-outs above the origin after D1 chromatography, as adopted for analysis of I-compounds, is potentially applicable. However, initial purification by reverse-phase TLC, followed by in situ transfer and resolution by PEI-cellulose TLC has been found to be most effective for these adducts. Reverse-phase TLC at 4 degrees C or in a stronger salt solution further improves retention of some adducts (e.g., mitomycin C and diaziquone adducts). For adduct separation by PEI-cellulose TLC, salt solutions with or without urea are used.
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PMID:32P-postlabelling analysis of small aromatic and of bulky non-aromatic DNA adducts. 822 92

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.
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PMID:Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics. 1033 10

The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.
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PMID:Effect of salts on the stability and folding of staphylococcal nuclease. 1132 80

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.
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PMID:Engineering the structural stability and functional properties of the GI domain into the intrinsically unfolded GII domain of the yeast linker histone Hho1p. 1587 77

We cloned complementary DNAs for 4SNc-Tudor protein (SN4TDR) from yellowtail (Seriola quinqueradiata), torafugu (Takifugu rubripes), and zebrafish (Danio rerio). This protein contains 4 staphylococcal nuclease domains at the N terminus followed by a Tudor domain. We also identified the 4SNc-Tudor proteins highly homologous to that in yellowtail from the Takifugu genomic database. According to the smart database, these fish proteins had an overlapping Tudor domain (smart00333) with a complete 5 SNc domain (smart00318). In addition, 2 possible translation start sites were observed at the 5' sequences in all 3 fish species. Northern blot analysis of different yellowtail organs showed that the full SN4TDR messenger RNA was approximately 4000 nucleotides long and that its expression was highest in liver and gallbladder, being about 2 to 5 times higher than in kidney, brain, ovary, and gills, and exceedingly low in spleen, heart, and muscle. A minor 2000-nucleotide transcript observed in kidney, spleen, and gallbladder, was attributable to an alternatively spliced variant of this gene. Total proteins extracted from yellowtail liver were fractionated by heparin affinity column chromatography and separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Analyses by SDS-PAGE and liquid chromatography with tandem mass spectroscopy identified the polypeptide encoded by SN4TDR as a single molecule of 102 kDa.
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PMID:Structural analysis of cDNAs coding for 4SNc-Tudor domain protein from fish and their expression in yellowtail organs. 1613 64

The reaction of polyuridylic acid with sodium bisulfite produces modified polymers in which up to 95% of the uracil residues have been converted to uracil-6-sulfonate residues. A 91.6% bisulfite-saturated polymer was found to resist hydrolysis by spleen phosphodiesterase and phosphorolysis by polynucleotide phosphorylase. Digestion by pancreatic ribonuclease was successful and gave the bisulfite adduct of uridine-3'-phosphate. Treatment of this nucleotide adduct with acid phosphatase afforded the bisulfite adduct of uridine. The ability of polyuridylic acid to bind to ribosomes, and to stimulate the binding of phenylalanine tRNA to ribosomes was abolished by progressive bisulfite saturation of the polymer. The rate of decline of these functionsf with increasing bisulfite content, was less sharp than the loss of phenyl-alanine coding ability o, the modified polymer.
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PMID:Modification of polyuridylic acid by bisulfite. II: Studies on ribosomal binding and enzymatic hydrolysis. 2419 77

We have analyzed Mytilus galloprovincialis' sperm chromatin, which consists of three protamine-like proteins, PL-II, PL-III, and PL-IV, in addition to a residual amount of the four core histones. We have probed the structure of this sperm chromatin through digestion with micrococcal nuclease (MNase) in combination with salt fractionation. Furthermore, we used the electrophoretic mobility shift assay to define DNA-binding mode of PL-II and PL-III and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. Although in literature it is reported that M. galloprovincialis' sperm chromatin lacks nucleosomal organization, our results obtained by MNase digestion suggest the existence of a likely unusual organization, in which there would be a more accessible location of PL-II/PL-IV when compared with PL-III and core histones. So, we hypothesize that in M. galloprovincialis' sperm chromatin organization DNA is wrapped around a PL-III protein core and core histones and PL-II and PL-IV are bound to the flanking DNA regions (similarly to somatic histone H1). Furthermore, we propose that PL's K/R ratio affects their DNA-binding mode and self-association ability as reported previously for somatic and sperm H1 histones.
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PMID:New insights into protamine-like component organization in Mytilus galloprovincialis' sperm chromatin. 2549 11


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