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)

We report high-resolution 13C and 15N NMR spectra of crystalline staphylococcal nuclease (Nase) complexed to thymidine 3',5'-diphosphate and Ca2+. High sensitivity and resolution are obtained by applying solid-state NMR techniques--high power proton decoupling and cross-polarization magic angle sample spinning (CPMASS)--to protein samples that have been efficiently synthesized and labeled by an overproducing strain of Escherichia coli. A comparison of CPMASS and solution spectra of Nase labeled with either [methyl-13C]methionine or [15N]valine shows that the chemical shifts in the crystalline and solution states are virtually identical. This result is strong evidence that the protein conformations in the solution and crystalline states are nearly the same. Because of the close correspondence of the crystal and solution chemical shifts, sequential assignments obtained in solution apply to the crystal spectra. It should therefore be possible to study the molecular structure and dynamics of many sequentially assigned atomic sites in Nase crystals. Similar experiments are applicable to the growing number of proteins that can be obtained from efficient expression systems.
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PMID:Comparison of the solution and crystal structures of staphylococcal nuclease with 13C and 15N chemical shifts used as structural fingerprints. 341 1

We have reported the presence of insulin-related poly A+ RNA sequences in human placenta by RNA to DNA hybridization. In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA. Poly A+ RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and [35S]methionine as a label. From 2.0 X 10(6) cpm of specifically incorporated [35S]methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14,000 represented about 0.1% of total radioactivity in the translational products of poly A+ RNA of first trimester placenta. A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs. This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation. Translational products synthesized from mRNA of term placenta showed a second labeled band of 24,000 daltons. This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain. A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis. Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation.
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PMID:Synthesis of somatomedin C/insulin-like growth factor I by human placenta. 354 54

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.
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PMID:Experimental methyl mercury neurotoxicity: locus of mercurial inhibition of brain protein synthesis in vivo and in vitro. 384 56

The reversible effect of dietary methionine deficiency was studied in young adult rats. The sensitivity of nuclear chromatin to micrococcal nuclease (EC3.1.4.7) digestion and the composition of the chromatin proteins were unaffected by the dietary regimens. The specific chromatin-bound RNA polymerase II activity decreased during methionine deficiency. Refeeding of methionine for 2 days restored the activity in the nuclease-released chromatin. RNA polymerase I plus III activity remained unchanged. Total RNA polymerase activity changed with the liver wet weight which was reduced during methionine deficiency and was not restored to control level after 2 days of methionine refeeding. RNA polymerase activity was altered by methionine deficiency. The recovery was independent of major modifications of the chromatin structure and protein composition.
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PMID:RNA polymerase activities and chromatin protein composition of rat liver during methionine deprivation and refeeding. 400 43

An analog of staphylococcal nuclease has been prepared in which all amino acids, except the six following, are fully deuterated: tryptophan; methionine; tyrosine, in ring positions 2 and 6; histidine, in ring position 2; aspartic acid and asparagine, beta-methylene; and glutamic acid and glutamine, gamma-methylene. The analog has a much simpler high-resolution nuclear magnetic resonance spectrum than the fully protonated enzyme. The effects of calcium ion and of the inhibitor 3', 5'-thymidine diphosphate on the spectrum of the analog were readily detected.
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PMID:High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. 567 35

To examine the distribution of 5-methylcytosine in chromatin DNA, DNA of HeLa cells was labeled with [3H-methyl]methionine and [14C] thymidine and analyzed after extensive digestion of the nuclei with micrococcal nuclease. When the chromatin solubilized with the nuclease was fractionated on a sucrose density gradient, DNA in mononucleosomes was considerably depleted in 5-methylcytosine, as compared with polynucleosomes. Electrophoretic separation of DNA from the chromatin also revealed the depletion of 5-methylcytosine in the mononucleosomal size of DNA. This was confirmed by the chromatographic analysis of 5-methyldeoxycytidine after enzymatic digestion of the DNA to nucleosides. Thus the DNA in mononucleosomes solubilized by extensive micrococcal nuclease digestion is depleted in 5-methylcytosine, suggesting that 5-methylcytosine is preferentially missing from the DNA in the nucleosome core particles.
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PMID:Undermethylation of DNA in mononucleosomes solubilized by micrococcal nuclease digestion of HeLa cell nuclei. 648 45

The mRNA species encoded by early region 4 (E4) (map position [mp] 91.5 to 99.3) of adenovirus 2 were isolated from the polysomes of infected KB cells and were purified by hybridization to the cloned HindIII-F fragment (mp 89.5 to 97.3) or to EcoRI-C fragment (mp 89.7 to 100). The mRNA's were translated in vitro using [35S]methionine as a labeled precursor in rabbit reticulocyte lysates treated with micrococcal nuclease as well as in wheat germ lysates. Five major (35,000-molecular-weight [35K], 23K, 22K, 21K, 18K) polypeptides were observed when the reticulocyte lysate was used. The 23K, 22K, 21K, and 18K polypeptides were also observed with the wheat germ lysate, as well as a very prominent 11K polypeptide; the 35K polypeptide was not observed. Assignment of these polypeptides to E4 was further established by hybrid arrested translation. Two-dimensional gel electrophoresis of a wheat germ translate resolved five polypeptides ranging from 18K to 23K, the major 11K polypeptide, and polypeptides of 10K and 9K. The in vitro 23K to 18K and 11K polypeptides migrated to approximately the same positions on two-dimensional gels as did seven 26K to 21K polypeptides and an 11K polypeptide synthesized in vivo (Brackmann et al., J. Biol. Chem, 255:6772--6779, 1980). Two-dimensional tryptic peptide maps demonstrated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related. The peptide map of 11K is different from those of the above polypeptides, although 11K may share one tryptic methionine polypeptide with them. These results indicate that E4 encodes a major 11K polypeptide, as well as major 35K, 23K, 22K, 21K, and 18K polypeptides.
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PMID:Identification of adenovirus 2 early region 4 polypeptides by in vitro translation and tryptic peptide map analysis. 708 56

As part of a study of the role of non-histone proteins in chromosome structure, the synthesis of non-histones associated with interphase chromatin was investigated. Synchronized suspension cultures of HeLa cells were pulse-labeled with [35S]methionine, and chromatin was prepared by mild micrococcal nuclease digestion. Two-dimensional polyacrylamide gel electrophoresis, in addition to one-dimensional electrophoresis, was used to resolve the patterns of incorporation of radioactive label. Significant variations in non-histone synthesis were seen during the cell cycle. A strong correlation was not found between DNA synthesis in mid-S phase and variations in non-histone synthesis. The non-histone proteins of purified metaphase chromosomes were also characterized by two-dimensional gel electrophoresis and compared to the proteins of interphase chromatin. The pattern of non-histones is not identical with that of interphase chromatin, although a number of major species may be shared by interphase chromatin and metaphase chromosomes. The HeLa nuclear scaffold, the framework that maintains the overall morphology of the interphase nucleus, shows relatively few proteins on two-dimensional gels. The synthesis of nuclear scaffold proteins was quantitated by excising each of 19 proteins from two-dimensional gels and determining the incorporated radioactivity by scintillation counting. Substantial variations in protein synthesis were found, with several species showing changes of about 2-fold in the percentage of incorporation.
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PMID:Role of non-histones in chromosome structure. Cell cycle variations in protein synthesis. 709 52

A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described. The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions. [35S]methionine incorporation was linear for 2 h. Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products. When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation. In immunoprecipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.
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PMID:Cell-free coupling of Newcastle disease virus RNA transcription, translation and Co-translational processing. 709 52

Saccharomyces cerevisiae general regulatory factor CP1 (encoded by the gene CEP1) is required for optimal chromosome segregation and methionine prototrophy. MET16-CYC1-lacZ reporter constructs were used to show that MET16 5'-flanking DNA contains a CP1-dependent upstream activation sequence (UAS). Activity of the UAS required an intact CP1-binding site, and the effects of cis-acting mutations on CP1 binding and UAS activity correlated. In most respects, MET16-CYC1-lacZ reporter gene expression mirrored that of chromosomal MET16; however, the endogenous gene was found to be activated in response to amino acid starvation (general control). The latter mechanism was both GCN4 and CP1 dependent. MET25 was also found to be activated by GCN4, albeit weakly. More importantly, MET25 transcription was strongly CP1 dependent in gcn4 backgrounds. The modulation of MET gene expression by GCN4 can explain discrepancies in the literature regarding CP1 dependence of MET gene transcription. Lastly, micrococcal nuclease digestion and indirect end labeling were used to analyze the chromatin structure of the MET16 locus in wild-type and cep1 cells. The results indicated that CP1 plays no major role in configuring chromatin structure in this region, although localized CP1-specific differences in nuclease sensitivity were detected.
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PMID:Role of the Saccharomyces cerevisiae general regulatory factor CP1 in methionine biosynthetic gene transcription. 789 81


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