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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Two new RNase inhibitors, SaI14 (Mr, approximately 14,000) and SaI20 (Mr, approximately 20,000), were isolated and purified from a Streptomyces aureofaciens strain. The gene sai14, coding for SaI14 protein, was cloned and expressed in Escherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor from Bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.
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PMID:Isolation and purification of two novel streptomycete RNase inhibitors, SaI14 and SaI20, and cloning, sequencing, and expression in Escherichia coli of the gene coding for SaI14. 951 32

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

Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is widely distributed in body fluids and in various epithelia; its gene has been completely sequenced, but its function has long remained elusive. We have found that Zn alpha 2gp has RNase activity, comparable to onconase but two orders of magnitude less than RNase A. The RNase activity of Zn alpha 2gp is characterized by maxima in pH at 7.5, in ionic strength at 50 mM NaCl, and in temperature at 60 degreesC. It is strongly inhibited by ZnCl2, but unaffected by MgCl2. It is partially inactivated (down to 20%) by the placental RNase inhibitor. On synthetic polyribonucleotide substrates, the RNase activity of Zn alpha 2gp is specific for pyrimidine residues [poly(C) and poly(U) equally] and cleaves only single-stranded RNA. For onconase, it has been demonstrated that the RNase activity depends on pyroglutamic acid (pyr 1) as the N-terminus; Zn alpha 2gp also has pyr 1, while RNase A does not. Alignment of the amino acid sequences of Zn alpha 2gp and onconase or RNase A reveals only modest matches. Despite the more substantial overall structural homology of Zn alpha 2gp to class I major histocompatibility complex proteins, Zn alpha 2gp has not been proven to be associated with the immune response and, conversely, we could not detect RNase activity in six class I HLA heavy chains.
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PMID:Zinc-alpha 2-glycoprotein has ribonuclease activity. 967 22

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

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a Kd of 4 x 10(-15) M.
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PMID:Ribonuclease 4, an evolutionarily highly conserved member of the superfamily. 976 Sep 89

The angiogenic and other biological functions of the angiogenins, members of the pancreatic RNase superfamily of proteins, are reviewed in the context of their primary and tertiary structures. The ribonucleolytic activity and interactions with the placental ribonuclease inhibitor have seen much study in the last few years. The mechanism of the angiogenic activity of angiogenin has recently been postulated as involving multiple interactions with other proteins through specific regions on the molecular surface of angiogenin. These molecular partners include heparin, plasminogen, elastase, angiostatin, actin and most importantly a 170-kilodalton receptor on subconfluent endothelial cells. The existence of the latter receptor was established in conjunction with a mitogenic activity of angiogenin on subconfluent cells. The levels of angiogenin in various physiological and disease states are summarized, including various studies on pregnancy and angiogenin. Correlations are seen between states of enhanced angiogenesis and angiogenin levels. An overview of the relationship of angiogenin and the other RNases of the superfamily showed that their genes all are in relative close proximity on human chromosome 14. Examination of the many expressed sequence tags published in the public databanks, for angiogenin and the other RNases, revealed that angiogenin and RNase-4 (the most evolutionarily conserved RNase), share various identical 5'-untranslated regions on their sets of messenger RNAs, suggesting that their genes are in very close proximity on chromosome 14 and that they are products of differential splicing. This in turn suggests that, in both humans and mice, expression of these two proteins is under identical control, with obvious implications for their biological activities. The evolutionary history of the angiogenins is examined briefly on the basis of the protein sequences of the human, rabbit, pig, two bovine and four mouse angiogenins, and two mouse angiogenin pseudogene sequences. The discrepancy between the conventional requirement for conservatism in structure to allow multimolecule interactions, and the actual fast-changing sequence of the angiogenins, in concert with the wide-ranging activity even in birds, of human angiogenin, is discussed.
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PMID:The angiogenins. 976 Sep 90

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


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