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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63,000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity.
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PMID:Poly(A) polymerase from Vigna unguiculata seedlings. A bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. 255 12

We have developed and characterized cell-free systems active in translation from unfertilized eggs, 30-min zygotes and hatched blastulae of the sea urchin Strongylocentrotus purpuratus. The ion concentrations selected for preparation of the lysates were 150 mM-K+, 40 mM-Na+, 40 mM-Cl-, 5 x 10(-7) M free Ca2+ and 1 mM free Mg2+. It was necessary to include the ribonuclease inhibitor RNas in the preparations to obtain full activity consistently. The pH optimum was 7.2 and was extremely sharp for the three S. purpuratus lysates. The temperature optima of the three lysates were remarkably similar to those of the intact unfertilized egg and embryos. Lysates from unfertilized egg and 30-min zygotes showed a temperature optimum at 15 degrees C. The hatched blastula lysate showed a broader temperature optimum with a shift to about 20 degrees C. The optimized lysates incorporated radiolabelled amino acids into polypeptides for up to 90 min. The polypeptides synthesized ranged in Mr from 200,000 to 20,000, suggesting that the mRNA in the lysates was intact and capable of directing the synthesis of complete polypeptides. Furthermore, the three lysates were capable of initiation, as demonstrated by inhibition of initiation using the inhibitors edeine and 7-methylguanosine 5'-triphosphate (m7GTP). At 15 degrees C, the transit times for the three lysates were: unfertilized egg, 40 min; 30-min zygotes and hatched blastula lysates, 20 min. These transit times are similar to those of intact eggs and embryos, and significantly, reflect the two-fold increase in elongation rate seen following fertilization in intact embryos. Thus, these lysates display many features and characteristic responses typical of intact eggs and embryos, indicating that the lysates should be useful tools for the analysis of translation control in early embryogenesis.
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PMID:Characterization of translation systems in vitro from three developmental stages of Strongylocentrotus purpuratus. 270

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO2. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA(Glu), ATP, Mg2+, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[3H]glutamate or 1-[14C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[14C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the alpha subgroup of purple bacteria.
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PMID:Distribution of delta-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups. 278 25

Extracts of Rad+ and radiation-sensitive (rad) mutants of the yeast Saccharomyces cerevisiae were examined for total Mg2+-dependent alkaline deoxyribonuclease activity and the presence of a nuclease that crossreacts immunologically with an antiserum raised against an endo-exonuclease from Neurospora crassa, an enzyme exhibiting both deoxyribo- and ribonuclease activities. No significant differences were observed in total deoxyribonuclease activity between Rad+ and rad mutants. The antibody precipitable activity, however, was found to be 30%-40% of the total alkaline deoxyribonuclease activity in logarithmically growing Rad+ cells. Extracts of stationary phase cells were lacking in antibody precipitable activity. Using immunoblot methods, a 72 kDa crossreacting protein was identified from logarithmically growing cells that was absent from stationary phase cells. In all radiation-sensitive mutants examined, except rad52, at least 20% of total activity was precipitable. Extracts from logarithmically growing rad52 mutants, including a rad52::LEU2 insertion mutant, exhibited less than 10% of the Rad+ precipitable activity; however, some crossreacting material was detected. Although, the level of endo-exonuclease activity is influenced by the RAD52 gene, it is not the product of this gene. The total deoxyribonuclease and the antibody precipitable endo-exonuclease activities were also followed during meiosis. Unlike the Rad+ strain which had previously been shown to have increased levels of total and immunoprecipitable endo-exonuclease as cells underwent meiosis, the rad52 mutant exhibited no increases in either category of nuclease activity. Given the importance of the RAD52 gene in repair, recombination and mutagenesis, the endo-exonuclease may be a significant component of these processes.
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PMID:An endo-exonuclease activity of yeast that requires a functional RAD52 gene. 283 Apr 67

To facilitate further studies of flavivirus transcription, cell extraction methods and in vitro reaction conditions which increased West Nile virus (WNV) RNA-dependent RNA polymerase activity were determined. Subcellular fractions from WNV-infected BHK-21/W12 cells were characterized with regard to their protein and RNA content and in vitro polymerase activity. In both a cytoplasmic fraction, designated S1, and a fraction enriched for outer nuclear membranes, designated S2, seven virus-specific proteins, NS5 (96 kilodaltons [kDa]), NS3 (67 kDa), E (48 kDa), NS1 (47 kDa), ns4a (26 kDa), ns2a (17 kDa), and ns2b (14.5 kDa), were detected. The fractions also contained virus-specific RNA and cellular rRNA and mRNA. Polymerase activity in S1 and S2 fractions from WNV-infected cells was concentrated by pelleting and consisted of two types of enzyme activities: the WNV RNA-dependent RNA polymerase and terminal transferases of cellular origin. Enhanced levels of WNV polymerase activity were obtained from these cell fractions by altering several of the in vitro reaction conditions. Although Mg2+ was the divalent cation preferred by WNV polymerase, virus-specific in vitro transcription was detected at reduced levels when Mn2+ (0.05 or 0.5 mM) was present as the sole divalent cation. Product analysis revealed that the viral polymerase incorporated radiolabeled ribonucleotides into three distinct RNA species. Free single-stranded genome-sized RNA which was LiCl insoluble and RNase sensitive was found by fingerprint analysis to have an oligonucleotide pattern similar to that of WNV genomic RNA. RNA molecules which comigrated as a broad band near the top of the gel were separable into LiCl-insoluble, partially RNase-sensitive replicative-intermediate RNA and LiCl-soluble, RNase-resistant replicative-form RNA. The cellular transferases added UMP or AMP residues to the 3'-termini of cellular mRNA, tRNA, and 18S and 28S rRNA. Although a cellular terminal transferase has been reported to function in initiation of poliovirus transcription, no labeling of the WNV RNA by either of these cellular enzymes was detected. Therefore, they appear to play no specific role in flavivirus RNA synthesis.
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PMID:Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. 302 63

Calcium-activated neutral protease (CANP) has been purified from the human placenta by chromatographic procedures. The purified enzyme is a heterodimer with one subunit of mol. wt 70 000 and another of mol.wt 32 000. It is a thiol protease, active at pH 7.5 at 30 degrees C in the presence of calcium. Half-maximal activation of the enzyme occurred with 800 microM Ca2+.Zn2+ (2 mM), ethylenediaminetetraacetic acid (EDTA)(5 mM) and ethyleneglycol-bis-N,N,N',N'-tetraacetic acid (EGTA)(2 mM) inhibited the enzyme, while Mg2+ (0.5 mM to 5 mM) had no effect on the enzyme in the presence of calcium. Mn2+ and Ca2+ activated the enzyme synergistically. CANP coexists with its endogenous inhibitor in the human placenta. The inhibitor is a protein, inactivated by trypsin and unaffected by RNase, DNase, acid and heat treatments; it inhibits the enzyme probably by interacting with the enzyme molecule itself rather than by sequestering calcium ions.
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PMID:Calcium-activated neutral protease from human placenta: purification and characterization. 308 82

The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated. Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues. The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex. A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme. Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity. Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase. Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase. Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation. The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem. We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.
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PMID:Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymatic probes. 309 57

Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.
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PMID:Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins. 310 66

The outer layer of the bacteriophage phi 6 nucleocapsid (NC) was removed by EDTA and reassociated with the core in the presence of Ca2+ or Mg2+. The core was relatively inaccessible to trypsin digestion, was composed of protein P1, and was in the dodecahedral framework reported previously. (H.T. Steely, Jr., and D. Lang, J. Virol. 51:479-483, 1984; Y. Yang and D. Lang, J. Virol. 51:484-488, 1984). The double-stranded RNA genome became RNase sensitive after EDTA treatment of the nucleocapsid.
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PMID:The dodecahedral framework of the bacteriophage phi 6 nucleocapsid is composed of protein P1. 311 Apr 30

In order to determine the mechanism and enzyme(s) responsible for 3' processing of tRNA precursors, we have developed an in vitro processing system that uses as substrates two SP6 RNA polymerase-generated transcripts of the gene for tRNA(Tyrsu3)+ that contain 49 extra 5'-nucleotides and either 5 or 25 extra 3'-nucleotides. A high speed supernatant fraction from an Escherichia coli strain deficient in five ribonucleases was found to accurately process both tRNA precursors in this system to the size of mature tRNA(Tyr). Final 3' end processing of each precursor occurs in an exonucleolytic manner to generate the correct 3' terminus; however, a prior endonucleolytic cleavage also is observed in processing of the longer precursor. The system requires Mg2+ and works optimally at about 50 mM KCl and pH 8-9. Dialysis of the supernatant fraction leads to loss of processing activity but can be restored to normal by the addition of inorganic phosphate or arsenate. Furthermore, nucleoside diphosphates are a product of the processing reaction. These data indicate that 3' processing in RNase-deficient extracts involves a phosphorolytic reaction. On the other hand, phosphate is not required for processing in RNase+ extracts, although it does aid in processing of the longer precursor. The usefulness of this in vitro system for studies of tRNA processing and the identity of the phosphate-requiring enzyme are discussed.
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PMID:3' processing of tRNA precursors in ribonuclease-deficient Escherichia coli. Development and characterization of an in vitro processing system and evidence for a phosphate requirement. 327 67


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