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)

Virion RNA of poliovirus type 1 has been analyzed for the linkage between genome-protein VPg and the polyribonucleotide chain. Hydrolysis of the linkage with acid or alkali and enzymatic degradation lead to the conclusion that the bond is neither a phosphodiester such as nucleotidyl-(P-O)-serine (or threonine) nor a phosphoramidate such as nucleotidyl-(P-N)-amino acid. VPg-RNA can be iodinated by the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] but not by the chloramine-T or lactoperoxidase procedures, an observation suggesting that VPg does not contain accessible tyrosine. However, VPg can be labeled with [3H]tyrosine in vivo. Hydrolysis of VPg-[32P]pUp with 5.6 M HCl at 110 degrees yielded 32P-labeled O4-(3'-phospho-5'-uridylyl)tyrosine that could be cleaved with micrococcal nuclease to O4-[32P]phosphotyrosine and uridine 3'-[32P]phosphate. These data establish that VPg is linked to the poliovirus genome by a bond between the O4 of tyrosine and the 5'-P atom of the terminal uridylic acid residue. The 5' end of polio genome RNA can now be described as VPg(Tyr-O)-pU-U-A-A-A-A-C-A-G.
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PMID:O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. 21 3

Administration of 17beta-estradiol to roosters induced the synthesis of vitellogenin in the liver. The mRNA that specifies this protein has been purified from the livers of estrogen-treated roosters and has been shown to have a molecular weight of 2.3 X 10(6) (Deeley, R.G., Gordon, J.I., Burns, A.T.H., Mullinix, K.P., Bina-Stein, M., and Goldberger R.F. (1977) J. Biol. Chem. 252, 8310-8319). In order to rigorously establish the identity of the polypeptide specified by this mRNA, we used a staphylococcal nuclease-treated, mRNA-dependent wheat germ cell-free translation system capable of synthesizing polypeptides as large as vitellogenin (monomer Mr = 240,000). Vitellogenin mRNA directs the in vitro synthesis of a polypeptide with the following features: (a) it co-migrates with authentic vitellogenin in SDS-polyacrylamide gels; (b) it is highly enriched for serine but is not phosphorylated; (c) it is immunoprecipitated by purified, monospecific, anti-vitellogenin antibody; and (d) it has an unusual cyanogen bromide cleavage pattern characteristic of vitellogenin. The most striking characteristic of the cyanogen bromide cleavage products is an extremely large polypeptide (Mr = 90,000) that contains two phosvitins. The kinetics of incorporation of serine and methionine into vitellogenin synthesized in the wheat germ cell-free translation system indicates that the phosvitins are located near the COOH-terminal portion of the molecule.
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PMID:In vitro translation of avian vitellogenin messenger RNA. 91 74

The Glu-43 residue of staphylococcal nuclease has been proposed to function as a general base that facilitates the attack of water on the phosphodiester substrate [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. With DNA as substrate, Vmax in the glutamate-43--serine (E43S) mutant enzyme is decreased by 2700-fold at pH 7.4 but only 376-fold at pH 9.9. With the wild-type enzyme, Vmax increases with pH to pH 9.2, above which it becomes less sensitive to further increase in pH, leveling off at pH 9.8. In contrast, Vmax of the E43S mutant continues to rise, first order in [OH-], to pH 9.8. Above pH 10 both activities fall irreversible. Hence the hydroxyl ion can partially replace the effect of Glu-43 on kcat, in accord with the proposed role of Glu-43 as a general base. The inflection point in the curve relating pH to log Vmax of the wild-type enzyme at pH 9.4 may reflect the ionization of a Ca2+-bound water, or of a Lys or Tyr residue at the active site. The activator Ca2+ and the competitive inhibitor Mn2+ bind to the E43S mutant an order of magnitude more weakly than to the wild-type enzyme as detected by kinetics and by direct metal binding studies, and approximately one additional water ligand on Mn2+ is found in the binary Mn2+ complex of the E43S mutant (1.4 +/- 0.2) as compared to that of the wild-type enzyme (0.8 +/- 0.2). These data suggest that Glu-43 coordinates the divalent cation in the binary enzyme-metal complex but dissociates from the metal to create a water binding site and to function as a general base in the ternary enzyme-metal-DNA complex. While a 2-fold weaker binding of DNA to the Ca2+ complex of the E43S mutant than to the wild-type enzyme is found by kinetic studies, an order of magnitude tighter binding of the competitive inhibitor 3',5'-pdTp to the Mn2+ and Ca2+ complexes of E43S is found by direct binding studies. Distances from Co2+ to phosphorus in the ternary enzyme-Co2+-pdTp complexes reveal coordination of only the 5'-phosphate by Co2+ on the wild-type enzyme but coordination of both the 3'- and 5'-phosphates of pdTp on the E43S mutant.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Kinetic and magnetic resonance studies of the glutamate-43 to serine mutant of staphylococcal nuclease. 256 22

The high-resolution X-ray crystal structure of staphylococcal nuclease suggests that the gamma-carboxylate group of Glu-43 is directly involved in catalysis as a general base that facilitates the attack of water on the substrate phosphodiester. We have used primer-directed, site-specific mutagenesis to generate aspartate, glutamine, asparagine, alanine, and serine substitutions for this residue. The Vmax/Km for the aspartate mutant is reduced 1400-fold and the values for the charge-neutral mutations are reduced 5000-fold relative to the wild-type enzyme. Although these reductions in catalytic efficiency might appear useful in quantitatively estimating the importance of general basic catalysis in the reaction catalyzed by the wild-type enzyme, the thermal stabilities and 1H NMR spectral properties of the mutants suggest that such interpretations are ambiguous. All five mutants have higher melting temperatures for thermal denaturation than the wild-type enzyme, suggesting that the mutants have enhanced thermal stabilities relative to the wild-type enzyme. Chemical shift changes relative to the wild type are observed in both the aromatic and upfield-shifted methyl group regions of the 1H NMR spectra of the aspartate and serine mutants, suggesting the presence of conformational differences between the wild-type and mutant enzymes. That these conformational differences may be large enough to be mechanistically relevant is suggested by comparisons of the magnitudes of nuclear Overhauser effect (NOE) correlations between the aromatic and upfield-shifted methyl group regions observed via two-dimensional nuclear Overhauser effect correlation spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutants of staphylococcal nuclease. Detection and localization by 1H NMR spectroscopy of conformational changes accompanying substitutions for glutamic acid-43. 289 75

Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients. Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long. Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments. The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second. Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes. A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones. These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions.
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PMID:A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex. 376 31

The mechanism by which changes in diet mediate levels of exportable enzymes and proenzymes in pancreatic tissue were studied in rats. The relative levels of mRNA coding for pancreatic amylase, lipase, procarboxypeptidases A and B, and the family of serine protease zymogens have been determined by the ability of isolated RNA to direct the synthesis of these products in a high-fidelity micrococcal nuclease-treated reticulocyte-lysate translation system. Translation products synthesized in vitro correlated directly with products synthesized in vivo in pancreatic lobules. Dietary adaptation was observed when dietary carbohydrate was increased from 0 to 58% at the expense of protein (81-23%). The increase in dietary carbohydrate over this range resulted in a 2-fold increase in amylase synthesis in pancreatic lobules and a 1.8-fold increase in mRNA-directed synthesis of amylase in the translation system in vitro. Concomitant with the decrease in dietary protein, synthesis of serine protease zymogens in pancreatic lobules and in the system in vitro decreased by approximately 50%. Over this range of dietary manipulation, ratios of amylase to serine proteases showed a 3.6-fold change. When dietary carbohydrate was further increased to 81% and protein reduced to 0, non-adaptive changes were observed since there was a decrease in amylase synthesis under conditions both in vivo and in vitro. mRNAs coding for pancreatic lipase and procarboxypeptidases A and B were unaffected by the dietary changes. These findings indicate that nutritional regulation in the tissue levels of pancreatic enzymes and proenzymes is mediated by changes in the content of active cytoplasmic mRNAs.
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PMID:Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas. 619 2

The phosphorylation of the proteins of heterogeneous nuclear ribonucleoprotein particles has been investigated in HeLa cells. 32Pi labeling of intact cells indicated that, of the six major particle proteins, the most heavily phosphorylated was the C1-protein (Mr = 42,000). This protein, together with C2 (Mr = 44,000), is also phosphorylated by [gamma-32P]ATP in particle extracts and in particles that are purified by sedimentation or exclusion chromatography. The C-proteins, together with their particle-associated kinase, were partially purified from isolated particles following dissociation with micrococcal nuclease. Proteins C1 and C2 co-purify on phosphocellulose chromatography, and their peak overlaps with that of a casein kinase activity. Evidence suggesting that this kinase activity is responsible for C-protein phosphorylation includes 1) the phosphorylation of C-proteins in the fractions where they overlap with the kinase, 2) the phosphorylation of added C-protein by fractions of the casein kinase which lack detectable C-protein, and 3) the similarities in catalytic properties of the casein kinase- and C-protein-phosphorylating activities. The purified kinase activity is cyclic nucleotide and Ca2+ independent. It is stimulated by polyamines, inhibited by heparin, and utilizes either GTP or ATP with high affinity. Serine residues are the major phosphate acceptors. These properties indicate that the kinase is casein kinase II or a closely related enzyme. Moreover, purified casein kinase II from rabbit liver effectively phosphorylates C-protein. These results suggest that C-proteins may be natural substrates for nuclear casein kinase II.
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PMID:Phosphorylation of the C-proteins of HeLa cell hnRNP particles. Involvement of a casein kinase II-type enzyme. 642 27

The phosphorylation of the high mobility group (HMG) proteins has been investigated in mouse Ehrlich ascites, L1210 and P388 leukemia cells, human colon carcinoma cells (HT-29), and Chinese hamster ovary cells. HMG 14 and 17, but not HMB 1 and 2, were phosphorylated in the nuclei of all cell lines with a serine being the site of modification for both proteins in Ehrlich ascites cells. Phosphorylation of HMG 14 and 17 was greatly reduced in cultured cells at plateau phase in comparison to log phase cells, suggesting that modification of HMG 14 and 17 is growth-associated. However, phosphorylation was not linked to DNA synthesis, since incorporation of 32P did not vary through G1 and S phase in synchronized Chinese hamster ovary cells. Treatment of HT-29 or Ehrlich ascites cells with sodium butyrate reduced HMG phosphorylation by 30 and 70%, respectively. The distribution of the phosphorylated HMG proteins in chromatin was examined using micrococcal nuclease and DNase I. 32P-HMG 14 and 17 were preferentially associated with micrococcal nuclease-sensitive regions as demonstrated by the release of a substantial fraction of the phosphorylated forms of these proteins under conditions which solubilized less than 3% of the DNA. Short digestions with DNase I did not show a marked release of 32P-HMG 14 or 17.
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PMID:The phosphorylation of high mobility group proteins 14 and 17 and their distribution in chromatin. 646 58

To investigate the role of protein p3 in bacteriophage phi 29 initiation of replication, we have studied the nature of the covalent linkage between protein p3 and phi 29 DNA. The protein-DNA compound was digested with micrococcal nuclease and pronase resulting in a nucleotidyl-peptide that was further digested by alkaline phosphatase and snake venom phosphodiesterase yielding 5'-dAMP. The DNA-protein linkage is sensitive to alkali. Treatment of the nucleotidyl-peptide with 0.1 M NaOH at 37 degrees C for 3 hr after phosphatase digestion released 5'-dAMP. Hydrolysis of the nucleotidyl-peptide with 5.8 M HCl at 110 degrees C for 90 min yielded O-phosphoserine. These results, together with the sensitivity of the DNA-protein linkage to snake venom phosphodiesterase and its resistance to hydroxylamine, indicate that protein p3 is covalently linked to phi 29 DNA through a phosphoester bond between L-serine and 5'-dAMP, namely a O,5'-deoxyadenylyl-L-serine bond.
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PMID:Protein p3 is linked to the DNA of phage phi 29 through a phosphoester bond between serine and 5'-dAMP. 677 79

Hydrogen bonds are a ubiquitous feature of protein structures, yet there is great uncertainty about the energetic contribution of hydrogen bonding to protein stability. This study addresses this question by making a series of single substitution mutations in the model protein staphylococcal nuclease. These mutants have had a residue capable of participating in hydrogen bonding either removed or introduced. The variants we have investigated are as follows: nine valines substituted with threonine and serine; eight threonines converted to valine, serine, and cysteine; and seven tyrosines replaced by phenylalanine and leucine. The stabilities of these 56 mutant proteins were determined by titration with guanidine hydrochloride using fluorescence as a probe of structure. In general, it was found that the stability effects of removing a hydrogen bonding residue and replacing it with a nonbonding residue were relatively small. This was true even in the case of buried residues participating in hydrogen bonds, where the substituted residue leaves an unfulfilled hydrogen bond in the hydrophobic core. In contrast, introducing a hydrogen bonding residue in place of a nonbonding residue was generally more costly energetically. A wide variability in the cost of burying a hydroxyl was observed, but this does not seem to be due to differences in hydrogen bonding. The overall energetic contribution of various wild-type hydrogen bonding interactions was evaluated as being favorable. A range of energies from approximately 1.5 to 4.0 kcal/mol was estimated for the contribution of these interactions to the stability of the native state.
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PMID:Energetic contribution of side chain hydrogen bonding to the stability of staphylococcal nuclease. 757 91


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