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)

Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion-exchange and reversed-phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cross-linked by three disulfide bonds, is glycosylated at Asn33, and is 57% identical to bovine angiogenin. The amino-terminal and carboxyl-terminal residues are pyroglutamic acid and proline, respectively. The protein has ribonucleolytic activity that is similar to, but somewhat lower than, that of bovine angiogenin, i.e. very low relative to RNase. It is angiogenically potent on chicken chorioallantoic membrane, but less so than angiogenin. The sequence and activities demonstrate that this protein is a second, distinct, member of the angiogenin sub-family of pancreatic ribonucleases, and is referred to as angiogenin-2.
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PMID:An angiogenic protein from bovine serum and milk--purification and primary structure of angiogenin-2. 926 95

mRNA stability is a limiting parameter for the efficiency of in vitro protein biosynthesis. In order to develop strategies to prolong the mRNA half-life, we investigated the ribonuclease activities in the complete Escherichia coli system, in the separate cell fractions 70S ribosomes and S-100 and in the non-cellular fraction. Our results imply that the amount of ribonucleolytic activities and the insensitivity to placental RNase inhibitor in the complete system are due to the 70S ribosome fraction, whereas the generation of small degradation products is due to the S-100 fraction. Remarkably, the human placental RNase inhibitor is able to reduce mRNA degradation in the bacterial S-100 fraction.
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PMID:Ribonucleolytic activities in the Escherichia coli in vitro translation system and in its separate components. 931 19

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
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PMID:Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties. 954 48

Significant amounts of ribonuclease inhibitor protein are present in human and rat erythrocytes, cells that are essentially devoid of ribonuclease or functional RNA. The protein from human erythrocytes is indistinguishable from human placental ribonuclease inhibitor protein by immunological and biochemical criteria. Each inhibitor forms an equimolar complex with bovine pancreatic ribonuclease A and is inactivated by treatment with the sulfhydryl reagent p-(hydroxymercuri)benzoate. Amino acid composition and several cycles of amino acid sequence analysis also showed apparent identify of the erythrocyte and placental proteins. We calculate a level of 1.5-3.5 x 10(4) molecules of active inhibitor per erythrocyte, most or all of which occurs in an uncomplexed form since inactivation of the inhibitor revealed barely detectable levels of RNase activity. Immunogold localization showed a high level of labeling and a uniform distribution of gold particles in the cytoplasm of erythrocytes, while little inhibitor activity was found in association with isolated red blood cell membranes. Oxidative stress on isolated red cells resulted in a decrease in the level of reduced glutathione and a gradual and irreversible loss of inhibitor activity; inhibitor disappeared from the cytosol and became associated with nascent Heinz bodies. We suggest a role for this protein in the metabolism and aging process of the erythrocyte.
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PMID:Ribonuclease inhibitor protein of human erythrocytes: characterization, loss of activity in response to oxidative stress, and association with Heinz bodies. 962 52

Eosinophil cationic protein (ECP) is one of two RNase A-superfamily ribonucleases found in secretory granules of human eosinophilic leukocytes. Although the physiologic function of eosinophils [and thus of the two eosinophil ribonucleases, ECP and eosinophil-derived neurotoxin (EDN)] remains controversial, we have recently shown that isolated human eosinophils promote ribonuclease-dependent toxicity toward extracellular virions of the single-stranded RNA virus, respiratory syncytial virus, group B (RSV-B). We have also shown that recombinant human EDN (rhEDN) can act alone as a ribonuclease-dependent antiviral agent. In this work, we provide a biochemical characterization of recombinant human ECP (rhECP) prepared in baculovirus, and demonstrate that rhECP also promotes ribonuclease-dependent antiviral activity. The rhECP described here is N-glycosylated, as is native ECP, and has approximately 100-fold more ribonuclease activity than non-glycosylated rhECP prepared in bacteria. The enzymatic activity of rhECP was sensitive to inhibition by placental ribonuclease inhibitor (RI). Although rhECP was not as effective as rhEDN at reducing viral infectivity (500 nM rhECP reduced infectivity of RSV-B approximately 6 fold; 500 nM rhEDN, >50 fold), the antiviral activity appears to be unique to the eosinophil ribonucleases; no reduction in infectivity was promoted by bovine RNase A, by the amphibian ribonuclease, onconase, nor by the closely-related human ribonuclease, RNase k6. Interestingly, combinations of rhEDN and rhECP did not result in either a synergistic or even an additive antiviral effect. Taken together, these results suggest that that the interaction between the eosinophil ribonucleases and the extracellular virions of RSV-B may be specific and saturable.
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PMID:Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. 964 19

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.
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PMID:Ribonuclease A variants with potent cytotoxic activity. 972 16

An enzyme-affinity-gold method to detect RNA in routinely prepared ultrastructural samples is based on the affinity of the gold-coupled enzyme, ribonuclease, for its substrate, RNA. High concentrations of a known inhibitor of RNase, heparin, are uniquely located in human mast cell granules. Specific labeling for the presence of heparin in these structures was determined using the RNase-gold (R-G) reagent based on the RNase inhibitor property of heparin. This property was used to probe for the presence of proteoglycans (PG) known to be present in a wide variety of ultrastructural samples, none of which contain heparin. In addition to known subcellular sites of RNA, the R-G reagent was shown to bind to PG-rich cytoplasmic granules in a wide variety of leukocytes and secretory cells of epithelial, endocrine, and neuroendocrine origin. This newly recognized property was used to image the changing distribution of labeled PGs during cellular maturation, secretion, and recovery from secretion of secretory cells in vivo, ex vivo, in vitro and in isolated, biochemically defined guinea pig basophil granule preparations.
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PMID:Ribonuclease-gold labels proteoglycan-containing cytoplasmic granules and ribonucleic acid-containing organelles--a survey. 1021 22

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.
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PMID:A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action. 1270 57

Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase.
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PMID:Insertional-fusion of basic fibroblast growth factor endowed ribonuclease 1 with enhanced cytotoxicity by steric blockade of inhibitor interaction. 1519 17

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. The experiment demonstrated that it might effectively inhibit tumor-induced angiogenesis and inhibit tumor growth. Ribonuclease inhibitor is constructed almost entirely of leucine-rich repeats, which might be involved in unknown biological effects besides inhibiting RNase A and angiogenin activities. The exact molecular mechanism of antitumor on ribonuclease inhibitor remains unclear so far. In order to further understand the function of ribonuclease inhibitor and investigate the relationship with tumor growth, our study established a transfection of human ribonuclease inhibitor cDNA into the murine B16 cells by the retroviral packaging cell line PA317. The cell line transfected with a stably high expression of ribonuclease inhibitor was identified. We found that the transfected ribonuclease inhibitor could obviously inhibit cell proliferation, regulate cell cycle and induce cell apoptosis in vitro. Mice that were injected with the B16 cells transfected RI cDNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower density of microvessels, longer latent periods, and survival time than those in the other two control groups. In conclusion, the results reveal the novel mechanism that antitumor effect of ribonuclease inhibitor is also associated with inducing apoptosis, regulating cell cycle and inhibiting proliferation besides antiangiogenesis. These results suggest that ribonuclease inhibitor might be a candidate of tumor suppressor gene in some tissues. RI could become a target gene for gene therapy. Our study may be of biological and clinical importance.
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PMID:Antitumor effects of human ribonuclease inhibitor gene transfected on B16 melanoma cells. 1577 86


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