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)

The mRNA present in extracts of mouse sarcoma 180 (S-180) ascites cells is relatively resistant to degradation when compared to added tracer ribosomal RNA. Deproteinized mRNA added to the extract is about as resistant as the endogenous mRNA, an indication that the protection is not due to any protein present in the endogenous mRNP structure. A major determinant of protection lies at the 5' end of RNA chains, where the presence of a triphosphate or a cap enhances the stability of mRNA transcripts. Addition of poly(A) to a capped transcript had little effect on stability. Stabilization by the cap structure is apparently not due to association of transcripts with a cap-binding protein. The discrimination in RNA decay rates appears to be based on interaction of the different RNA species with an exonuclease, which represents the predominant ribonuclease activity in the extract. Other major cytoplasmic nucleases are suppressed by an RNase inhibitor that is present in excess.
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PMID:Protection of mRNA against nucleases in cytoplasmic extracts of mouse sarcoma ascites cells. 848 53

Onconase, or P-30, is a protein initially purified from extracts of Rana pipiens oocytes and early embryos based upon its anticancer activity both in vitro and in vivo. It is a basic single-chain protein with an apparent molecular mass of 12,000 daltons and is homologous to RNase A. In cultured 9L glioma cells, onconase inhibits protein synthesis with an IC50 of about 10(-7) M. The inhibition of protein synthesis correlates with cell death determined by clonogenic assays. 125I-Labeled onconase binds to specific sites on cultured 9L glioma cells. Scatchard analysis of the binding data shows that onconase appears to bind to cells with two different affinities, one with a Kd of 6.2 x 10(-8) and another of 2.5 x 10(-7) M. Each cell could bind about 3 x 10(5) molecules of onconase at each of the two affinity sites. The low affinity Kd is similar to the IC50 for onconase toxicity. Onconase also demonstrates a saturability of cytotoxicity at a concentration that would saturate the low affinity binding site. Incubation at 4 degrees C increased the binding of onconase to cells relative to 37 degrees C binding and also increased the sensitivity of cells to onconase toxicity, indicating that receptor binding may be an initial step in cell toxicity. Onconase cytotoxicity can be blocked by metabolic inhibitors, NaN3 and 2-deoxyglucose, and cytotoxicity is potentiated 10-fold by monensin. Ribonuclease activity appears necessary for onconase toxicity because alkylated onconase, which only retains 2% of the ribonuclease activity, was at least 100-fold less potent in inhibiting protein synthesis in cells. Onconase inhibition of protein synthesis in 9L cells coincides with the degradation of cellular 28 S and 18 S rRNA. In contrast to RNase A, onconase is resistant to two RNase inhibitors, placental ribonuclease inhibitor and Inhibit-Ace. Northern hybridization with placental ribonuclease inhibitor cDNA probe indicates that 9L glioma cells contain endogenous placental ribonuclease inhibitor mRNA. Based on these results, we propose that onconase toxicity results from onconase binding to cell surface receptors, internalization to the cell cytosol where it degrades ribosomal RNA, inhibiting protein synthesis and causing cell death.
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PMID:A cytotoxic ribonuclease. Study of the mechanism of onconase cytotoxicity. 848 18

The gene for human angiogenin (Ang), a member of the ribonuclease superfamily, was fused to a gene encoding a single-chain antibody (sFv) against the human transferrin receptor. Three Ang single-chain immunofusion proteins (AngsFvs) were constructed with variations in the type of linker connecting the VL and VH chain [EGKSSGSGSESKEF, L1 or (GGGGS)3, L2] as well as with or without a spacer (FB) connecting the Ang and sFv (AngFBsFvL1 or L2; AngsFv(L2)]. Although the nature of the linker did not affect the enzymatic activity of the FB-containing fusion proteins, the fusion protein containing the L2 linker was 2.3-fold more effective than the L1 linker in competing with the labeled monoclonal IgG1 antibody for binding to the transferrin receptor. The fusion protein containing the L2 linker without the FB spacer exhibited a 13-fold decrease in binding to the transferrin receptor as well as a decrease in its capacity to degrade tRNA and to inhibit translation in the rabbit reticulocyte lysate compared to its counterpart containing the FB spacer. Binding of placental ribonuclease inhibitor (PRI) to Ang also was affected by the nature of the linker and by the presence or absence of a spacer. PRI bound to Ang and AngFBsFv(L2) and inhibited their ribonuclease activity. A 3-fold greater concentration of PRI, however, did not affect the activity of AngFBsFv(L1) or AngsFv(L2), suggesting that the conformation of these fusion proteins was altered. Binding of monoclonal and polyclonal anti-Ang antibodies to AngsFvs was also used to investigate conformational alterations of the fusion proteins. AngFBsFv(L2) was the least altered while AngFBsFv(L1) exhibited the greatest change in structure. Yet maximal concentrations of all AngsFvs elicited angiogenesis in the chick chorioallantoic membrane assay, demonstrating that Ang in all three fusion proteins remained functionally active. Consistent with all the activities, the fusion protein containing the FB spacer and L2 linker was the most cytotoxic to three different human tumor cell lines. The fusion protein lacking the FB spacer exhibited the least cytotoxicity. These data demonstrate that the linker connecting the VH-VL chains can affect the binding and cellular cytotoxicity of Ang immunofusions and that placement of a spacer between the antibody binding domains and Ang is necessary for optimal activity. Thus, a new class of targeted therapeutic agents containing Ang as the toxic moiety can be designed that potentially will be less immunogenic and less toxic than immunotoxins available currently.
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PMID:Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains. 855 26

The cytoplasm of mammalian cells of undoubtedly contain a number of different ribonuclease activities, few if any of which have been well characterized. We describe the purification of an exoribonuclease from rabbit reticulocytes which is able to degrade capped RNAs in a 5' to 3' manner. The purified enzyme contains polypeptides of 62 and 58 kDa and may contain an additional polypeptide of 54 kDa. It behaves as a complex of 150 kDa when analyzed by HPLC gel retardation on Superdex 200HR. It is heat-labile, dependent upon divalent cations (Mg2+) for activity, resistant to placental ribonuclease inhibitor, and active over a broad range (10-200 mM) of monovalent cation (K+) concentrations. The enzyme requires a polynucleotide chain of at least 10 bases for activity and cleaves oligonucleotides, up to an octamer long, from the 5' end of an appropriate substrate. In the case of a capped RNA substrate, product analysis by TLC and PAGE indicates that a capped trinucleotide or tetranucleotide or both is produced. Examination of the kinetics of the enzyme with capped and triphosphate-terminated substrates shows that that the cap structure inhibits the action of the enzyme. Furthermore, the data suggest that the rate-limiting step involves the positioning of the enzyme at the 5' end of the substrate and/or cleavage of the first internucleotide bond.
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PMID:Purification and characterization of a 5' to 3' exoribonuclease from rabbit reticulocytes that degrades capped and uncapped RNAs. 862 Aug 71

The effect of the protein RNase inhibitor (PRI) on the activity of bovine seminal RNase (BS-RNase) was investigated using the isolated quaternary forms, MxM and M=M, of the enzyme reported earlier [Piccoli, R., et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1870-1874]. We found that the inhibitor does not interact with the intact isolated forms but has dramatic, differential effects on the two forms when the assays are performed under reducing conditions. These conditions, which are essential for full activity of the inhibitor, and are typical of its cytosolic localization, also promote monomerization of the M=M form, while under identical conditions the MxM form becomes a noncovalent dimer (NCD). The sensitivity of BS-RNase or that of the isolated quaternary forms under reducing conditions thus appears to be related to differential monomerization of the two forms of the enzyme; monomer being sensitive to PRI. The present study also shows that the interconversion between the two forms in equilibrium occurs at much higher rates in a reducing environment and that PRI further affects the interconversion and alters the equilibrium favoring monomerization of the protein. An opposite effect on the equilibrium between the forms is played by the substrate, which is found to stabilize the NCD form of the protein with a shift in the equilibrium between the two forms towards the dimer. These results are analyzed in the light of the antitumor action of the enzyme which is exerted in the cytosol, i.e., in the compartment housing the PRI and the ribosomal RNA, the molecular target of the enzyme.
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PMID:Effects of protein RNase inhibitor and substrate on the quaternary structures of bovine seminal RNase. 867 17

Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNase inhibitor was conjugated to CNBr-activated Sepharose in the presence of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was eluted with 3 M NaCl at pH 5.0. Two 25-kDa and 18-kDa RNases, which were obtained from human liver using a cellulose phosphate column, were bound to the immobilized RNase inhibitor and recovered in a pure and active form after treatment of the resin with p-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with different sugar contents.
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PMID:Purification of mammalian ribonuclease using immobilized human ribonuclease inhibitor. 881 55

A simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8-10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory-type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as substrate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory-type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental-type RNase inhibitor, can be performed.
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PMID:pH gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: detection and inhibition of enzyme activity in the gel. 890 26

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.
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PMID:RT-PCR without RNA isolation. 896 38

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an Rcryst of 19.4% at 2.5 A resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the p1 phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 A2 of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (Ki = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer.
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PMID:Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A. 900 Jun 28

RNase inhibitor (RI) binds with extraordinary affinity (Ki approximately 10(-13)-10(-16) M) to diverse proteins in the pancreatic RNase superfamily. In the present study, the structural basis for the recognition of two RI ligands, human angiogenin (Ang) and bovine RNase A, has been investigated by site-specific mutagenesis of human RI and Ang. The RI residues examined were those that appear to form strong contacts with RNase A in the crystal structure of the porcine RI x RNase A complex [Kobe, B. & Deisenhofer, J. (1995) Nature (London) 374, 183-186] that would not be replicated in the Ang complex. Ala substitutions of five of these residues (Glu-287, Lys-320, Glu-401, Cys-408, and Arg-457) were found to have little or no effect on binding of RNase A. In contrast, replacements of Tyr-434, Asp-435, and Tyr-437 and deletion of the C-terminal residue Ser-460 substantially weakened affinity for RNase A: the losses of binding energy associated with the mutations were 5.9, 3.6, 2.6, and 3.5 kcal/mol, respectively. Thus these four residues, which are neighbors in the tertiary structure, appear to constitute a "hot spot" for the RNase A interaction. However, only one of them, Asp-435, was equally important for binding of Ang; the Ki increases produced by mutations of the others were 20- to 93-fold smaller for Ang than for RNase A. Consequently, Tyr-434 plays a significant but lesser role in the Ang complex, whereas Tyr-437 and Ser-460 make only minor contributions. Ala mutations of four Ang residues (His-8, Gln-12, Asn-68, and Glu-108) that correspond to RI contacts on RNase A produced no major changes in affinity for RI. These findings indicate that RI uses largely different interactions to achieve its extremely tight binding of RNase A and Ang.
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PMID:Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A. 905 Aug 52


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