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)

Human pancreatic ribonuclease 1 (hRNase 1) in the mature form has been produced in E. coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3',5'-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase 1 to the characteristics of the protein.
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PMID:Recombinant human pancreatic ribonuclease produced in E. coli: importance of the amino-terminal sequence. 748 19

The performance characteristics of a potent new RNase inhibitor have been evaluated, as have applications for its use in molecular biology. PRIME Inhibitor is a protein, not related to the commonly available human placental RNase inhibitors (HPRI). It has a high specific activity, enhanced temperature stability, broad reaction pH range and significantly greater cost-effectiveness than commercial HPRI. PRIME Inhibitor is suitable for use in in vitro transcription, in vitro translation, first- and second-strand cDNA synthesis, preparation of RNA and mRNA, and reverse transcription-polymerase chain reaction.
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PMID:A potent, cost-effective RNase inhibitor. 754 11

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

Ribonuclease inhibitor (RI) was purified about 1300-fold from human cerebrum (including a small portion of midbrain) by a combination of ammonium sulfate precipitation, ribonuclease A-Sepharose chromatography, and high-performance anion-exchange chromatography. The purified RI appeared to be homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using the same method, a homogeneous RI was also obtained from human hindbrain (brainstem and cerebellum). The cerebral RI appeared to be virtually identical with the hindbrain RI on the basis of the following properties: (a) Molecular mass was estimated to be 50 kDa on SDS-PAGE under reducing conditions. (b) Composition analysis revealed that the RI was rich in leucine and cysteine residues and included no amino sugars. (c) The N-terminus was blocked and probably modified by N-acetylation. After treatment with trifluoroacetic acid, it became susceptible to Edman degradation and was sequenced as Ser-Leu-Asp-Ile-Gln-Ser-Leu-Asp-Ile-Gln-(Cys)-Glu-Glu-. (d) The RI, which showed sulfhydryl-dependent inhibitory activity on both secretory-type and nonsecretory-type ribonucleases, bound tightly to ribonuclease to form a 1:1 complex on a molar basis. (e) The RI cross-reacted strongly with anti-human placental RI antibody. These findings also indicate that human brain RI is quite similar to human placental RI. In contrast to the abundance of RI in human brain tissue (about 0.08% (w/w) of total protein), RI was undetectable in human cerebrospinal fluid, suggesting that brain RI may not be a secreted protein.
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PMID:Purification and characterization of human brain ribonuclease inhibitor. 803 55

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.
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PMID:A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization. 805 28

The cytosolic, alkaline RNase in rat vaginal epithelial cells (VEC) from normal, immature rats was found to be present largely in the free, active form unlike in many other mammalian tissues where it is known to be present in a latent form as a complex with RNase inhibitor (RNasin). Estradiol (E2) administration induced expression of RNasin activity in the VEC from such animals and caused virtually total inhibition of cytosolic RNase activity in these cells by 12 h after the hormone injection. These changes may have metabolic implications in relation to other biochemical events stimulated by estradiol in rat VEC.
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PMID:Modulation of cytosolic RNase activity by endogenous RNase inhibitor in rat vaginal epithelial cells on estradiol administration. 816 9

Residues 287-301 of human placental ribonuclease inhibitor have been reported to inhibit pancreatic ribonuclease A in a similar way as the entire inhibitor (Crevel-Thieffry, I., Cotterill, S. and Schuller, E. (1992) Biochim. Biophys. Acta 1122, 107-112). Using three different assays, we were unable to observe inhibition by the synthetic peptide. Moreover, the peptide did not compete with the entire inhibitor for binding to RNAase A.
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PMID:Residues 287-301 of human ribonuclease inhibitor do not affect ribonuclease activity and inhibitor binding; a reply. 821 87

Ribonuclease inhibitor is a cytoplasmic protein that tightly binds and inhibits ribonucleases of the pancreatic ribonuclease superfamily. The primary sequence of this inhibitor contains leucine-rich repeats (LRRs); these motifs are present in many proteins that participate in protein-protein interactions and have different functions and cellular locations. In vivo, ribonuclease inhibitor may have a role in the regulation of RNA turnover in mammalian cells and in angiogenesis. To define the structural features of LRR proteins and to understand better the nature of the tight interaction of ribonuclease inhibitor with ribonucleases, we have determined the crystal structure of the porcine inhibitor. To our knowledge, this is the first three-dimensional structure of a protein containing LRRs and represents a new class of alpha/beta protein fold. Individual repeats constitute beta-alpha structural units that probably also occur in other proteins containing LRRs. The non-globular shape of the structure and the exposed face of the parallel beta-sheet may explain why LRRs are used to achieve strong protein-protein interactions. A possible ribonuclease-binding region incorporates the surface formed by the parallel beta-sheet and the beta alpha loops.
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PMID:Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. 826 99

Interleukin 2 (IL2) mRNA has a short half-life in the cytoplasm of T lymphocytes, relative to most mRNA. We have discovered a candidate ribonuclease to account for the rapid turnover of IL2 mRNA in the cytosol of the human T lymphocyte cell line Jurkat. In partially purified form, this RNase is about 7 times as active on IL2 as on beta-globin mRNA. Pancreatic RNase, by contrast, does not show a significant preference for IL2 mRNA. Neither 5' capping, nor polyadenylation of the substrate mRNAs affects their degradation by the IL2-selective mRNase, whose activity is optimal in 0.5 mM Mg++ and 100 mM potassium acetate. The mRNase behaves like a protein of molecular weight 60-70,000 on gel chromatography, and is unusual in that it is insensitive to placental RNase inhibitor (RNasin). The mRNase cleaves IL2 mRNA at a small number of sites in the coding region, and IL2 mRNA containing only the coding region and 36 nucleotides of the 3'-noncoding region competes efficiently with full-length IL2 mRNA for the mRNase, whereas beta-globin mRNA does not.
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PMID:An RNasin-resistant ribonuclease selective for interleukin 2 mRNA. 844 10

Sialic acid-binding lectin (SBL) isolated from Rana catesbeiana eggs is a basic protein which agglutinates a large variety of tumour cells and has an amino acid sequence homologous to that of human angiogenin and pancreatic ribonuclease (RNase). Although SBL and angiogenin lack the Cys-65-Cys-72 disulphide bond of pancreatic RNase, the locations of the other three disulphide bonds are similar among the three molecules. SBL was found to exhibit RNase activity, as well as catalytic properties resembling those of bovine RNase A in some respects. For example, SBL hydrolyses poly(uridylic acid) and poly(cytidylic acid) as substrates, and prefers the former. RNase A and angiogenin are strongly inhibited by human placental RNase inhibitor, whereas the RNase activity and tumour cell agglutination activity of SBL are not affected by this inhibitor.
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PMID:Ribonuclease activity of sialic acid-binding lectin from Rana catesbeiana eggs. 844 85


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