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)

The thyroid hormone-responsive protein (THRP) is expressed in rat cerebral tissue and has 83% overall sequence homology with c-Abl interactor protein, Abi-2, which is a substrate for the tyrosine kinase activity of c-Abl. Within the core region of the two proteins, the sequence similarity approaches 99%. To determine whether THRP is a rat homologue of Abi-2 or is a distinct protein with unique properties, the tissue distribution of THRP and Abi-2 mRNA's was examined using a sensitive ribonuclease protection assay and probes specific for THRP and Abi-2, respectively. The THRP mRNA content of cerebral tissue (1340.0 +/- 126.5 arbitrary units) was 2.3-fold higher than Abi-2 mRNA (581.3 +/- 73.7), while the ratio of hepatic content of THRP mRNA (209.0 +/- 49.1) to hepatic Abi-2 mRNA (2923.0 +/- 378.7) was only 0.07 (P < 0.004). Very low levels of Abi-2 mRNA, but not THRP mRNA, were also found in the heart and small intestine. Experiments with PC12 cells transfected with the full-length THRP cDNA and grown in the presence or absence of a tyrosine kinase inhibitor, along with experiments where PC12 cells were cotransfected with the THRP cDNA with or without the wild-type or mutant (tyrosine kinase deficient) c-Abl cDNA, showed that THRP is tyrosine phosphorylated; however, it is not a substrate for c-Abl. These studies demonstrate that THRP and Abi-2 have distinct tissue distribution and distinct biological properties.
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PMID:Partial characterization of a cerebral thyroid hormone-responsive protein. 1188 98

By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)*GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from -11.7 kcal/mol for Val-tRNA(Glu) to -8.1 kcal/mol for Val-tRNA(Tyr), clearly establishing EF-Tu*GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of k(off) varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism.
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PMID:The tRNA specificity of Thermus thermophilus EF-Tu. 1189 Dec 93

Using radioimmuno- and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24 h treatment, sGnRH ([Trp(7),Leu(8)]-LHRH) and sGnRH-A ([D-Arg(6),Pro(9)]-LHRH) (0.1 nM-1 microM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10 nM) on GH secretion was observed after 6 h incubation, while no action on the GH mRNA level were noted until 12 h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II ([His(5),Tyr(8)]-LHRH), and finally LHRH and LHRH-A([D-Trp(6), Pro(9)]-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.
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PMID:Effects of gonadotropin-releasing hormone on growth hormone secretion and gene expression in common carp pituitary. 1203 58

In sequel to our preliminary observations with peptidomimetic opioid compounds, we have further investigated immunomodulatory activity of one peptidomimetic compound (Tyr-NH-CH2-CH2-O-Phe-NH2) with peripheral blood mononuclear cells (PBMCs) of healthy volunteers/tuberculosis patients. This peptidomimetic compound was evaluated for its effect on purified protein derivative (PPD) stimulated lymphocyte proliferation in vitro, production of Th1 and Th2 cytokines by ELISA and ribonuclease protection assay. Our study shows the immunosuppressive potential of above synthetic peptidomimetic compound. This compound inhibited PPD stimulated human lymphocyte proliferation and this inhibition was reversed by opioid receptor antagonist, naloxone. Its immunosuppressive effect was further demonstrated by inhibition of interleukin-9 (IL-9), IL-10 but failed to influence IL-2, IL-15 and interferon-y (IFN-gamma) in PPD stimulated human PBMCs.
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PMID:Inhibition of antigen specific lymphocyte proliferation and cytokine stimulation by peptidomimetic opioid compound. 1209 65

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.
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PMID:Two distinct regions on the surface of an RNA-binding domain are crucial for RNase E function. 1242 3

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.
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PMID:Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2). 1252 9

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond. The reductive unfolding pattern of onconase is consistent with an analysis of the exposed surface area of the cysteine sulfur atoms in the (30-75) disulfide bond, which reveals that these atoms are about four- and sevenfold, respectively, more exposed than those in the next two maximally exposed disulfides. By contrast, in the reductive unfolding of the homologue, RNase A, there are two intermediates, arising from the reduction of the (40-95) and (65-72) disulfide bonds, which takes place in parallel, and on a much longer time-scale, compared to the initial reduction of onconase; this behavior is consistent with the almost equally exposed surface areas of the cysteine sulfur atoms that form the (40-95) and (65-72) disulfide bonds in RNase A and the fourfold more exposed cysteine sulfur atoms of the (30-75) disulfide bond in onconase. Analysis and in silico mutation of the residues around the (40-95) disulfide bond in RNase A, which is analogous to the (30-75) disulfide bond of onconase, reveal that the side-chain of tyrosine 92 of RNase A, a highly conserved residue among mammalian pancreatic ribonucleases, lies atop the (40-95) disulfide bond, resulting in a shielding of the corresponding sulfur atoms from the solvent; such burial of the (30-75) sulfur atoms is absent from onconase, due to the replacement of Tyr92 by Arg73, which is situated away from the (30-75) disulfide bond and into the solvent, resulting in the large exposed surface-area of the cysteine sulfur atoms forming this bond. Removal of Tyr92 from RNase A resulted in the relatively rapid reduction of the mutant to form a single intermediate (des [40-95] Y92A), i.e. it resulted in an onconase-like reductive unfolding behavior. The reduction of the P93A mutant of RNase A proceeds through a single intermediate, the des [40-95] P93A species, as in onconase. Although mutation of Pro93 to Ala does not increase the exposed surface area of the (40-95) cysteine sulfur atoms, structural analysis of the mutant reveals that there is greater flexibility in the (40-95) disulfide bond compared to the (65-72) disulfide bond that may make the (40-95) disulfide bond much easier to expose, consistent with the reductive unfolding pathway and kinetics of P93A. Mutation of Tyr92 to Phe92 in RNase A has no effect on its reductive unfolding pathway, suggesting that the hydrogen bond between the hydroxyl group of Tyr92 and the carbonyl group of Lys37 has no impact on the local unfolding free energy required to expose the (40-95) disulfide bond. Thus, these data shed light on the differences between the reductive unfolding pathways of the two homologous proteins and provide a structural basis for the origin of this difference.
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PMID:Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues. 1509 46

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.
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PMID:Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase. 1521 23

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.
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PMID:Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes. 1532 60

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


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