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)

A ribonuclease that preferentially cleaves natural and synthetic double-stranded (ds) RNAs has been partially purified from Pennisetum typhoides. The enzyme degrades [3H]poly(rA) X poly(rU) to acid-soluble products. The ds RNase does not degrade RNA:DNA hybrid but appears to have about 18% activity against single-stranded (ss) RNAs under the assay conditions used for the cleavage of ds RNAs. The RNase has a molecular weight of 35,000 daltons as determined from gel filtration using Sephadex G-200. The ds RNase shows an absolute requirement for divalent cations Mg++ or Mn++ and monovalent cations K+ or Na+. The specificity of the enzyme towards ds RNA template is supported by the inhibition of cleavage of ds RNAs by ethidium bromide and Penicillium chrysogenum viral ds RNA. The enzyme preparation acts on ds RNAs isolated from Saccharomyces cerevisiae and P. chrysogenum virus. The purified ds RNase also cleaves the in vitro transcriptional products of adenovirus DNA and this activity is inhibited by 5 mM ethidium bromide suggesting that ds regions of adenovirus RNA are involved in the cleavage process.
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PMID:Action of Pennisetum typhoides double-stranded ribonuclease on viral ds RNAs. 391 48

This paper reviews briefly work carried out in our laboratory on the relative roles of the macrophage plasma membrane receptor (CR3) for the cleaved third complement component (iC3b) and the mannosyl/fucosyl receptor (MFR) in binding, ingestion and respiratory burst (RB) response elicited by promastigotes versus amastigotes of Leishmania donovani. In the absence of serum soluble inhibitors (mannan, ribonuclease B) of the MFR cause a dose-dependent reduction in the numbers of promastigotes binding to murine resident peritoneal macrophages and in the proportion of bound parasites eliciting a RB response. For amastigotes no consistent reduction in binding in the presence of mannan is observed but the proportion of parasites eliciting a RB is reduced. Serum-independent binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, is also inhibited by the anti-CR3 monoclonal antibody M1/70, by Fab anti-C3, and by an inhibitor of C3 fixation, sodium salicyl hydroxamate. For amastigotes, which are poor activators of the alternative pathway, a lesser effect is observed with all three inhibitors of CR3-mediated binding. The results obtained with these three independent inhibitors provide strong evidence that cleaved macrophage-derived C3 (iC3b), which can be visualised on the parasite surface in electron microscope sections following addition of anti-C3 antibody and a protein A-gold conjugate, mediates binding to CR3. Modulation experiments in which either CR3 or MFR are rendered inaccessible demonstrate that both receptors must be present on the segment of the macrophage membrane with which the parasite makes contact to mediate binding and ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of macrophage complement and lectin-like receptors in binding Leishmania parasites to host macrophages. 391 May 69

Sterile, concentrated culture supernatant from Pasteurella haemolytica (biotype A, serotype 1) strain 630 was subjected to physical, chemical, and immunologic treatments to determine their influence on leukotoxin (cytotoxin) activity contained in the supernatant. Each treated sample contained approximately 8 chemiluminescence inhibitory units of leukotoxin. Treatment effects were evaluated for their ability to inactivate leukotoxin activity. Leukotoxin activity in treated samples was determined by inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. Optimal leukotoxin synthesis by P haemolytica occurred when the bacteria were at the logarithmic growth phase, whereas stationary phase cultures contained minimal amounts of leukotoxin activity in their culture supernatant. Leukotoxin activity was heat labile; activity was substantially decreased when concentrated culture supernatant samples containing leukotoxin activity were incubated at 37 C for several hours. When concentrated culture supernatant was incubated at progressively decreasing temperatures, there was a progressive increase in the length of time that the leukotoxin retained its biologic activity. Samples stored at -70 C retained activity for at least 2 months. Leukotoxin activity was nondialyzable and was able to withstand considerable extremes in hydrogen ion concentration. Leukotoxin activity could not be pelleted when subjected to forces of 100,000 X g for 1 hour. Chemical and enzymatic studies suggested that P haemolytica leukotoxin contained carbohydrate and protein moieties. Chemical treatment with 0.2% sodium lauryl sulfate, 0.5% sodium deoxycholate, 7.5 mM EDTA and 8M urea with 8 mM 2-mercaptoethanol and enzymatic treatment with lipase, ribonuclease, and deoxyribonuclease had no discernible effect on leukotoxin activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pasteurella haemolytica leukotoxin: physicochemical characteristics and susceptibility of leukotoxin to enzymatic treatment. 396 75

A ribonuclease that hydrolyzes either linear duplex or single-stranded RNA in an exonucleolytic manner has been partially purified from Ehrlich ascites tumor cell nucleoli and is free from other ribonucleases. The enzyme will also degrade the RNA complement of an RNA X DNA duplex; however, no nuclease activity is observed on linear duplex or single-stranded DNA. The exonuclease acts on RNA nonprocessively from the 3' end releasing 5'-mononucleotides. The enzyme has a broad pH optimum around pH 8.0, requires Mg2+ or Mn2+ (0.06 mM) for optimum activity, and is sensitive to ethylenediaminetetraacetic acid and N-ethylmaleimide inhibition. Monovalent cations including K+, Na+, and NH4+ are inhibitory. Gel filtration studies of this enzyme gave a Stokes radius of 40 A. Sedimentation velocity measurements in glycerol gradients yield a S20,W of 6.0 S. From these values a native molecular weight of 100 000 was calculated. Copurification of the single- and double-stranded activities, identical reaction requirements, and identical heat-inactivation curves strongly suggest that both activities reside with the same enzyme.
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PMID:Purification and properties of a novel nucleolar exoribonuclease capable of degrading both single-stranded and double-stranded RNA. 399 80

A rapid method for the extraction and purification of DNA from human leukocytes was developed. Crude nucleic acids were obtained by sodium dodecylsulfate (SDS) lysis and potassium acetate precipitation of other cellular material, and the DNA was purified by ribonuclease digestion, diethylaminoethyl (DEAE) cellulose chromatography and ethanol precipitation. DNA obtained by this method is biologically active as reflected by its ability to act as substrate for various nucleases and T4 DNA ligase. The yield was sufficiently high that DNA from less than 1 ml of blood could be used for a number of reactions.
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PMID:A rapid method for the extraction and purification of DNA from human leukocytes. 399 5

Native disulphide-bonded prolactin (band III) was distinguished from reduced prolactin (band II) and intermediate unstable disulphide-linked conformations by: (a) faster mobility of the former in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and (b) high-pressure liquid chromatography analyses of tryptic-digested peptides derived from prolactin in various conformations during its refolding pathway from reduced, unfolded to native conformation. The electrophoretic separation has been used to examine the state of disulphide bonding in newly synthesised prolactin translated from bovine pituitary mRNA in a rabbit reticulocyte translation system supplemented with nuclease-treated dog pancreatic microsomal membranes. The formation of correct disulphide pairing in prolactin (band III), synthesised in the in vitro translation system in the presence of pancreatic microsomes, required the presence of a thiol oxidant such as oxidised glutathione during the translation. The action of thiol oxidants on the in vitro biosynthesised and microsomally processed prolactin were both dose-dependent and catalytic; non-thiol oxidants such as NAD+ and NADP+ were ineffective. Examination of the time course of addition of oxidised glutathione to translating lysates showed that efficient and correct disulphide pairing in newly biosynthesised prolactin occurred when the oxidant was present co-translationally, but much lower yields of correctly disulphide-bonded prolactin were obtained when the oxidant was added after translation and processing were complete. The presence of protein-disulphide isomerase in dog pancreatic microsomes, employed in the in vitro translation system to process preprolactin, was demonstrated by (a) two-dimensional polyacrylamide gel electrophoresis of the membrane proteins, and (b) enzymic activity to accelerate reactivation of scrambled ribonuclease. Protein-disulphide isomerase activity was latent in intact microsomal vesicles, full activity being expressed upon sonication. A procedure has been devised to prepare pancreatic microsomal vesicles depleted of protein-disulphide isomerase which are active in processing and segregating in vitro biosynthesised prolactin. These membranes in the presence of low concentrations of oxidised glutathione are less active but in the presence of saturating levels of oxidised glutathione are fully competent in forming correct disulphide bridges in newly synthesised prolactin.
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PMID:Studies on the formation of intrachain disulphide bonds in newly biosynthesised bovine prolactin. Role of protein-disulphide isomerase. 406 47

The means by which coxsackievirus type A9 (CA9) is inactivated by proteolytic enzymes was investigated. After reaction of (14)C-leucine-labeled CA9 with Pronase, free leucine was liberated as measured by radiochromatography. Treatment of (14)C-leucine-labeled CA9 with trypsin or proteolytic filtrates of Pseudomonas aeruginosa caused the release of a variety of labeled substances. The extent of viral ribonucleic acid (RNA) release after exposure of CA9 to Pronase was determined by RNA infectivity tests or trichloroacetic acid solubility tests. Infective viral RNA was found not to be consistently released by reaction of CA9 with Pronase, but further treatment with 1% sodium dodecyl sulfate at pH 7.0 promoted viral RNA release. Sodium dodecyl sulfate treatment of CA9 that had not been reacted with Pronase did not inactivate virus or cause viral RNA release. Reaction of Pronase with (32)P-labeled CA9 resulted in the liberation of virus components soluble in cold trichloroacetic acid, whereas untreated CA9 or CA9 reacted with ribonuclease were precipitated by cold trichloroacetic acid. These results demonstrate that the primary means by which protease-sensitive enteroviruses are inactivated is by degradation of the virus capsid, with subsequent release of viral RNA.
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PMID:Degradation of coxsackievirus type A9 by proteolytic enzymes. 420 58

1. Treatment of washed rat liver microsomes in a medium containing 0.12m-sucrose, 12.5mm-potassium chloride, 2.5mm-magnesium chloride and 25mm-tris-hydrochloric acid buffer, pH7.6, with 2m-lithium chloride at 5 degrees for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting (14)C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH-2,6-dichlorophenol-indophenol reductase, NADH-neo-tetrazolium reductase, NADH-cytochrome c reductase and ribonuclease activities. 5. (3)H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of (3)H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.
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PMID:Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride. 431 14

After the attachment of radioactive coxsackievirus B3 to HeLa cells at 0 C and subsequent incubation at 37 C, 50 to 80% of attached virus radioactivity was eluted from the cells within 1 hr. Eluted virus had a buoyant density of 1.21 in a potassium tartrate gradient, sedimented more slowly than native virus in sucrose gradients, was resistant to ribonuclease, was unstable in CsCl centrifugation, and did not reattach to uninfected cells. Electrophoretic studies of sodium dodecyl sulfate-disrupted B3 virus in sodium dodecyl sulfate-polyacrylamide gels revealed four radioactive virus polypeptides (VP 1 to 4), of which the three largest migrated slightly faster than their poliovirus T1 counterparts. In contrast, electrophoretic analysis of eluted virus, after banding in a tartrate gradient or pelleting by centrifugation, showed the absence of the fastest migrating polypeptide, VP 4. VP 4 was recovered in the supernatant fluid when the eluted virions were removed by high-speed centrifugation. The results indicate that VP 4 is located at the surface of the native virion, and its dissociation from the capsid may represent the first specific alteration of the virion after virus-receptor interaction at the cell surface.
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PMID:Specific alterations of coxsackievirus B3 eluted from HeLa cells. 433 54

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.
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PMID:Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium. 433 82


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