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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine. Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1. Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type. In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.
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PMID:Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity. 1114 47

Escherichia coli cells were irradiated with ultraviolet light to stop ribosomal RNA synthesis. After infection of such cells with the single-stranded RNA phage R17, the RNA most rapidly labeled by a pulse of tritiated uridine sedimented in a broad band in the 16S region of sucrose gradients. The effect of ribonuclease on this material and its behavior during a "chase" period in nonradioactive medium suggest that it consists of a core of double-stranded RNA, one strand of which-the viral strand-is continually displaced by a growing strand forming single-stranded tails and ultimately free 27S viral RNA.
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PMID:REPLICATION OF THE RNA OF BACTERIOPHAGE R17. 1419 Feb 42

A new ultrasensitive differential scanning calorimeter (DSC) instrument is described, which utilizes autosampling for continuous operation. High scanning rates to 250 deg/h with rapid cooling and equilibration between scans facilitates higher sample throughput up to 50 samples during each 24 h of unattended operation. The instrument is suited for those pharmaceutical applications where higher throughput is important, such as screening drug candidates for binding constant or screening solution conditions for stability of liquid protein formulations. Results are presented on the binding of five different anionic inhibitors to ribonuclease A, which included cytidine 2'-monophosphate (2'CMP), 3'CMP, uridine 3'-monophosphate, pyrophosphate, and phosphate. Binding constants K(B) (or dissociation constants K(d)) are obtained from the shift in the transition temperature T(M) for ribonuclease thermal unfolding in the presence of ligand relative to the transition temperature in the absence of ligand. Measured binding constants ranged from 155 M(-1) (K(d) = 6.45 mM) for the weak-binding phosphate anion to 13100 M(-1) (K(d) = 76.3 microM) for the strongest binding ligand, 2'CMP. The DSC method for measuring binding constants can also be extended to ultratight interactions involving either ligand-protein or protein-protein binding.
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PMID:An autosampling differential scanning calorimeter instrument for studying molecular interactions. 1509 Jan 59

Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.
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PMID:Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay. 1555 7

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.
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PMID:Accumulation and turnover of 23S ribosomal RNA in azithromycin-inhibited ribonuclease mutant strains of Escherichia coli. 1609 36

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with RNase activity. The purpose of this study was to produce an active E(rns) for further applications using the yeast secreted expression system. The E(rns) gene was cloned into the expression vector pGAPZalphaC which was introduced into Pichia pastoris. Expression of E(rns) protein in culture supernatant was confirmed by Western blot analysis using both the monoclonal antibody against CSFV E(rns) and CSFV-positive swine serum. The yeast-expressed E(rns) (yE(rns)) was shown to have N-linked glycosylation and to form homodimer of 74 kDa molecules. All monomer, homodimer, and deglycosylated forms of yE(rns) demonstrated intrinsic ribonuclease activity and a clear preference for uridine-rich sequence. A direct sandwich blocking enzyme-linked immunosorbent assay (ELISA) based on the yE(rns) was developed with a high sensitivity and specificity. The yE(rns) which possesses enzymatic activity and retains antigenicity may provide a useful material for developing a diagnostic kit.
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PMID:Secreted expression of the classical swine fever virus glycoprotein E(rns) in yeast and application to a sandwich blocking ELISA. 1621

Gibberellic acid (GA) enhances the synthesis of alpha-amylase and ribonuclease in isolated aleurone layers and this process is inhibited by abscisin. Removal of gibberellic acid in mid-course of alpha-amylase production results in a slowing down of alpha-amylase synthesis, suggesting a continued requirement of GA for enzyme synthesis. This is paralleled by a continuous requirement for RNA synthesis. Addition of 6-methylpurine or 8-azaguanine in mid-course results in an inhibition of alpha-amylase synthesis within 3 to 4 hours. However, actinomycin D added in mid-course is almost without effect. This is not due to its failure to enter the cells, because it does inhibit (14)C-uridine incorporation at this stage. Addition of abscisin to aleurone layers which are synthesizing alpha-amylase results in an inhibition of this synthesis within 2 to 3 hours. Cycloheximide on the other hand inhibits enzyme synthesis immediately upon its addition. These data are consistent with the hypothesis that the expression of the GA effect requires the synthesis of enzyme-specific RNA molecules. The similarity in the kinetics of inhibition between abscisin on the one hand and 8-azaguanine or 6-methylpurine on the other suggests that abscisin may exert its action by inhibiting the synthesis of these enzyme-specific RNA molecules or by preventing their incorporation into an active enzyme-synthesising unit.
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PMID:Hormonal control of enzyme synthesis: on the mode of action of gibberellic Acid and abscisin in aleurone layers of barley. 1665 90

Protoplasts isolated from Avena sativa L. leaves undergo progressive senescence when incubated aseptically in 0.6 m mannitol with or without added nutrients. This senescence is manifested by morphological deterioration and ultimate lysis of protoplasts, by a decrease in incorporation of [(3)H]uridine and [(3)H]leucine into macromolecules, and by a sharp increase in ribonuclease activity.The presence in the incubation medium of l-arginine, l-lysine, certain polyamines related to these amino acids (cadaverine, putrescine, spermidine), Ca(2+), or streptomycin stabilizes the protoplasts. Protoplasts incubated with 10 mml-arginine or l-lysine show an initial inhibition of [(3)H]uridine incorporation, but with time, incorporation is restored to levels greater than in control protoplasts. The rise in ribonuclease activity of protoplasts is completely inhibited if the protoplasts are incubated with 10 mml-arginine. Greater incorporation of [(3)H]uridine into RNA of aging protoplasts is also maintained by appropriate concentration of cadaverine, putrescine, spermidine, Ca(2+), or streptomycin in the incubation medium; the same concentrations of these substances stabilize the protoplasts against additional lysis.
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PMID:Stabilization of Oat Leaf Protoplasts through Polyamine-mediated Inhibition of Senescence. 1666 Jan 39

The distribution of incorporated synthetic cytokinins (N(6)-[8-(14)C]benzyladenine ([8-(14)C]bzl(6)Ade) and N(6)[8-(14)C]furfuryladenine ([8-(14)C]fr(6)Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-(14)C]bzl(6)Ade or [8-(14)C]fr(6)Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-(14)C]bzl(6)A or [8-(14)C]fr(6)A in the rRNA peak after the fractionations. [8-(14)C]fr(6)A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.The distribution of [8-(14)C]fr(6)A moieties in mono- and oligonucleotides derived from combined treatment of [8-(14)C]fr(6)Ade-labeled rRNA with ribonuclease T(1) and pancreatic ribonuclease A was measured by fractionating the digest on a DEAE-cellulose column in the presence of 7 m urea and determining the [8-(14)C]fr(6)A content in each fraction. The [8-(14)C]fr(6)A content in the oligonucleotides varied from 46 to 210 mumol/mol of adenosine (A). The mol ratio of [8-(14)C]fr(6)A to A was three to four times greater for oligonucleotides containing uridine or cytidine ([A](n)Up or [A](n)Cp) than for those containing quanosine ([A](n)Gp).
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PMID:Distribution of incorporated, synthetic cytokinins in ribosomal RNA preparations from tobacco callus. 1666 Feb 83

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin although not by the ribonuclease inhibitor SUPERase* In. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development.
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PMID:Characterization of the ribonuclease activity on the skin surface. 1673 88


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