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)

A bacteriocin produced by Bacteroides fragilis 1356 was purified from culture medium and characterized. The spectrum of the inhibitory activity of this bacteriocin was species specific. The bacteriocin was recovered from the initial stages of purification as a complex, greater than 2 X 10(7) daltons in mass, containing protein, lipid, and carbohydrate. The dissociation of this complex by 6.0 M guanidine hydrochloride permitted further purification of the bacteriocin by removal of lipid and carbohydrate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified bacteriocin was homogeneous, with a relative molecular weight of 5,000. The activity of the purified bacteriocin was not affected by RNase, DNase, phospholipase A, pancreatic lipase, or dextranase, but was destroyed by trypsin, proteinase K, heat (80 degrees C, 30 min), or a pH below 5 or above 8. Amino acid analysis indicated a predominance of acidic and polar amino acids.
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PMID:Purification and characterization of a bacteriocin from Bacteroides fragilis. 635 Feb 64

Bovine retinas contain a factor that stimulates proliferation of aortic endothelial cells in culture as well as neovascularization on the chicken chorioallantoic membrane. The stimulatory activity has been partially purified from a balanced salt solution extract of bovine retinas. The stability of the activity to acid pH was utilized as the first step in purification. The acid-treated material was subjected to ion-exchange chromatography on DEAE Bio-Gel A. The material that was eluted with 0.1 M NaCl/50 mM Tris.HCl (pH 7.6) stimulated both the proliferation of vascular endothelial cells in culture and angiogenesis in vivo. A molecular weight of between 50,000 and 100,000 for the native molecule is suggested by ultrafiltration and gel chromatography; gel electrophoresis of partially purified material under reducing and denaturing conditions revealed the presence of two major components with apparent molecular weights of 50,000 and 70,000. The endothelial cell stimulatory activity was stable to pH 4-9 and to heating at up to 60 degrees C for 30 min, to incubating for 1 hr with DNase or RNase, to incubating for 2 hr with immobilized Pronase or immobilized or soluble trypsin, and to treating with 1 mM 2-mercaptoethanol, 4 M urea, or 2 M guanidine. Heating at 65-75 degrees C for 30 min, boiling for 2 min, extreme acidic (pH 2) or basic (pH 12) conditions, treating with 0.02% NaDodSO4, or incubating (5 hr) with soluble Pronase destroyed the activity. The significance of an angiogenic factor derived from retina stems from the fact that neovascularization is a serious complication in a number of ocular diseases.
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PMID:Angiogenic activity from bovine retina: partial purification and characterization. 694 16

Spirosomes, very find spiral particles, were isolated from a protoplastlysate of Lactobacillus brevis ATCC 8287 by differential centrifugation and purified further by potassium tartrate density gradient centrifugation. The purified spirosome preparation showed a maximum peak around 275 nm on the ultraviolet absorption spectrum and it consisted of about 94.5% protein. The buoyant density in CsCl of the spirosomes was 1.320 g/cm3. The spirosomes were composed mainly of a single protein (spirosin with an apparent molecular weight of about 95,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The protein of the spirosomes was found to be composed predominantly of neutral amino acids accompanied by approximately equal amounts of acidic and basic amino acids. The spirosomes showed one antigenic determinant in the immunodiffusion test. The spirosomes were readily degraded by the action or proteolytic enzymes and lost their antigenicity, but they were not affected by treatment with either deoxyribonuclease or ribonuclease. The spiral structure of the spirosome was also found to be disintegrated by treatment with 1 M guanidine hydrochloride, 4 M urea or 0.1% SDS, but not by the action of deoxycholate, nonionic detergents or mercaptoethanol, as observed in the electron microscope.
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PMID:Purification and characterization of spirosomes in Lactobacillus brevis. 710 79

The degradation of S--S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S--S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S--S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S--S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S--S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S--S bonds of some other proteins. The initial product of S--S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins.
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PMID:Degradation of protein disulphide bonds in dilute alkali. 721 43

The application of the PCR to complex samples is hindered by amplification inhibitors. We describe a reverse transcription-PCR-based method capable of inhibitor removal for the detection of enteroviruses in shellfish. Initial virus extraction stages based on a modified polyethylene glycol precipitation technique (G.D. Lewis and T.G. Metcalf, Appl. Environ. Microbiol. 54:1983-1988, 1988) were followed by virus purification with 1,1,2-trichloro,2,2,1-trifluoroethane and concentration by ultrafiltration. A guanidine isothiocyanate-glass powder extraction system was utilized for sample lysis, RNase protection, and nucleic acid purification. Removal of PCR inhibitors and method sensitivity were quantified in shellfish (oysters and mussels) seeded with poliovirus. PCR sample tolerance exceeded 4 g for depurated shellfish; however, polluted field samples were more inhibitory. Virus recoveries of 31% for oyster extracts and 17% for mussel extracts and nucleic acid extraction reverse transcription-PCR detection limits down to 1 PFU yielded an overall sensitivity limit of < 10 PFU of poliovirus in up to 5 g of shellfish. PCR-positive results were obtained from a variety of polluted field samples naturally contaminated with human enteroviruses. The methods developed for virus recovery and PCR inhibitor removal should be equally applicable to detection of other RNA viruses such as hepatitis A virus, Norwalk virus, and other small round-structured viruses in shellfish.
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PMID:Development of a method for detection of enteroviruses in shellfish by PCR with poliovirus as a model. 752 97

Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.
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PMID:Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo. 767 57

We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.
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PMID:Simple method for extracting RNA from cultured cells and tissue with guanidine salts. 768 4

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.
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PMID:Stability of ribonuclease T2 from Aspergillus oryzae. 779 25

A novel noncollagenous protein of the mineralized matrix of bovine bone was isolated by ion exchange and gel permeation chromatography. The apparent M(r) of the protein is 63,000 as determined by SDS-polyacrylamide gel electrophoresis. The protein is a rather minor constituent in bone and could not be detected in other connective tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. The 63-kDa protein was detected in the osteoid and around the osteocytes upon immuno-histochemical staining of bovine compact bone. The sequence of the 63-kDa protein was deduced from cDNA clones isolated from a rat calvaria lambda gt11 expression library. The protein contains two centrally located EF-hand Ca(2+)-binding domains. Seven heptad repeats are present indicating the ability of the protein for coiled-coil interactions. Ability to bind calcium was confirmed by 45Ca2+ binding to protein blotted onto nitrocellulose membrane. The protein was synthesized in calvaria explants as detected by immunoprecipitation of radiolabeled protein from the culture medium. Although the protein can be detected in biochemical amounts in bone only, varying amounts of mRNA for this protein were detected in several rat tissues by RNase protection assay with highest levels in rat calvaria. This extracellular protein corresponds to a mouse protein called nucleobindin.
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PMID:Isolation, characterization, and primary structure of a calcium-binding 63-kDa bone protein. 789 Jul 46

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.
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PMID:Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions. 800 82


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