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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In recent years, the enzymes which are involved in the formation of DHT in steroid target tissues have been well investigated, however, enzymes responsible for the catabolism and elimination of steroids in these tissues, in particular the uridine diphospho-glucuronosyltransferase (UGT) family of enzymes, have received much less attention. We have recently demonstrated that human and monkey are unique in having high plasma levels of C19 steroid glucuronides. These circulating conjugates have been proposed to reflect the peripheral conversion of adrenal and gonadal C19 steroids to potent androgens, especially DHT. In humans, the presence of steroid UGT activities is found in the liver and several extrahepatic tissues including the prostate, mammary gland and ovary. In addition, UGT activities were observed in breast and prostate tumor cell lines such as MCF-7 and LNCaP, respectively. In agreement with the presence of steroid conjugating enzymes in extrahepatic tissues, UGT cDNA clones, which encode steroid conjugating proteins, have been isolated from libraries constructed from human and monkey prostate mRNA. The presence of UGT transcripts and proteins in extrahepatic tissues in both species, as determined by Northern blot, ribonuclease protection, specific RT-PCR, in situ hybridization, Western blot and immunocytochemistry analysis, indicate the relevance of steroid glucuronidation in tissues other than the liver. Knowing that both the human prostate and the human prostate cancer LNCaP cell line express steroid metabolizing proteins, including UGT enzymes, regulation of UGT mRNA and protein levels, as well as promoter activity was studied in these cells. The results demonstrate a differential regulation between the two highly related isoforms UGT2B15 and UGT2B17, where only the expression of UGT2B17 was affected following treatments of LNCaP cells with androgens, growth factors or cytokines. Steroid conjugation by UGT enzymes is potentially involved in hormone inactivation in steroid target tissues, thus modifications in UGT expression levels may influence hormonal responses.
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PMID:Characterization of UDP-glucuronosyltransferases active on steroid hormones. 1041 20

The reactions of a ribonuclease model substrate, the compound uridine-3'-p-nitrophenyl phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis. The cyclization of this compound is subject to catalysis by general base (by imidazole buffer), specific base (by carbonate buffer), and by acid. All three reactions proceed by the same mechanistic sequence, via cyclization to cUMP, which is stable under basic conditions but which is rapidly hydrolyzed to a mixture of 2'- and 3'-UMP under acid conditions. The isotope effects indicate that the specific base-catalyzed reaction exhibits an earlier transition state with respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction. Stereochemical analysis indicates that both of the base-catalyzed reactions proceed with the same stereochemical outcome. It is concluded that the difference in the nucleophile in the two base-catalyzed reactions results in a difference in the transition state structure but both reactions are most likely concerted, with no phosphorane intermediate. The (15)N isotope effects were also measured for the reaction of the substrate with ribonuclease A. The results indicate that considerably less negative charge develops on the leaving group in the transition state than for the general base-catalyzed reaction in solution. Copyright 2000 Academic Press.
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PMID:Kinetic Isotope Effects and Stereochemical Studies on a Ribonuclease Model: Hydrolysis Reactions of Uridine 3'-Nitrophenyl Phosphate. 1091 50

Pentavalent organo-vanadates have been used extensively to mimic the transition state of phosphoryl group transfer reactions. Here, decavanadate (V(10)O(28)6-) is shown to be an inhibitor of catalysis by bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry shows that the Kd for the RNase A decavanadate complex is 1.4 microM. This value is consistent with kinetic measurements of the inhibition of enzymatic catalysis. The interaction between RNase A and decavanadate has a coulombic component, as the affinity for decavanadate is diminished by NaCl and binding is weaker to variant enzymes in which one (K41A RNase A) or three (K7A/R10A/K66A RNase A) of the cationic residues near the active site have been replaced with alanine. Decavanadate is thus the first oxometalate to be identified as an inhibitor of catalysis by a ribonuclease. Surprisingly, decavanadate binds to RNase A with an affinity similar to that of the pentavalent organo-vanadate, uridine 2',3'-cyclic vanadate.
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PMID:Decavanadate inhibits catalysis by ribonuclease A. 1101 16

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


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