Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fluorescence properties of the Y base of yeast tRNA-Phe are known to be quite sensitive to the environment. The fluorescence lifetime of the Y base in yeast tRNA-Phe is identical in orthorhombic crystals and in the mother liquor from which these crystals are grown. It is 10% higher than the lifetime observed in dilute solutions of tRNA. This small change is a solvent effect due to isopropyl alcohol in the crystallization medium. Isopropyl alcohol does not change the accessibility of the chromophore of the Y base as measured by iodide quenching rates in solution. The accessibility in intact tRNA-Phe is much less than in a ribonuclease digest. Thus, within the limits of the sensitivity of the method, the Y chromophore occupies the same environment in solution and in the crystal and it must be at least partially buried.
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PMID:A comparison of the fluorescence of the Y base of yeast tRNA-Phe in solution and in crystals. 109 57

1. In a previous report we described three isozymes of intracellular ribonuclease in Dictyostelium discoideum, which were found in vegetative cells. Here we report that the molecular weights of the three isozymes from vegetative cells. 2. They are 14.3 kDa, 60 kDa and 80 kDa, as determined by activity-staining of gels after SDS-PAGE. 3. For renaturation of ribonucleolytic activity from D. discoideum cells after SDS-PAGE, fibrinogen-containing gels were used and gels were washed in aqueous isopropanol to remove detergent. Results of studies by this method suggest that each of these isozymes is composed of only a single polypeptide. 4. The effect of the buffer system on this technique is discussed.
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PMID:Determination of the molecular weights of ribonuclease isozymes in a cell-free crude extract of Dictyostelium discoideum, by activity-staining of gels after SDS-PAGE. 137 16

The interactions of tryptophan-59 (TRP-59) and its protein environment in ribonuclease-T1 (RNAse-T1) were examined in a 50-ps molecular dynamics simulation. The simulation used was previously shown to demonstrate a fluorescence anisotropy decay that closely agreed with the experimentally determined limiting anisotropy for RNAse-T1 (Axelsen, P. H., C. Haydock, and F. G. Prendergast. 1988. Biophys. J. 54:249-258). Further characterization of TRP-59 side chain dynamics and its protein environment has now been completed and correlated to other photophysical properties of this protein. Angular fluctuations of the side chain occur at rates of 1-10 cycles/ps and are limited to +/- 0.3 radians in all directions. Side chain motions are primarily limited by nonpolar collisions, although most side chain atoms have some collisional contact with polar atoms in the adjacent protein matrix or water. The steric relationship between PRO-39 and TRP-59 changes abruptly at 16 ps into the simulation. Two types of interaction with water are observed. First, a structural water appears to H-bond with the greater than N-H group of TRP-59. Second, water frequently contacts the six-atom ring. The electrostatic field experienced by the TRP-59 rings appears to be relatively constant and featureless regardless of ring orientation. We make the following interferences from our data: The fluorescent emission of TRP-59 may be red-shifted relative to TRP in nonpolar solvents either as a result of specific interactions with the structural water or relaxations of proximal bulk water and polar protein moieties. The quenching efficiency of polar interactions with TRP-59 must be extremely low given their frequency and the high quantum yield of RNAse-T1. This low efficiency may be due to restricted and unfavorable interaction geometries. PRO-39 is located near two titratable HIS residues in RNAse-T1 and may be involved in pH-dependent fluorescence phenomena by virtue of a metastable interaction with TRP-59. The interaction of bulk water with TRP-59 illustrates features of the gated transition state model for transient exposure to exogenously added collisional quenching agents. The restrictive environment of TRP-59 suggests that extrinsic quenching can only occur via interactions with the edge of the indole six-atom ring and that the efficiency of a quencher in a protein environment is likely to be a function of molecular symmetry.
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PMID:Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence. 250 98

A methodological study of practical importance to protein sequencing has been carried out. Peptide mapping and sequence analysis of the cleavage products of reduced and carboxymethylated ribonuclease have been applied to the study of the activity and specificity of trypsin, chymotrypsin, elastase, lysyl endopeptidase (Achromobacter protease I), endoproteinase Arg-C (from mouse submaxillary gland), Staphylococcus aureus V8 protease, pepsin, and thermolysin in the presence of 20% methanol, ethanol, 2-propanol, and acetonitrile at 22 and 37 degrees C. The peptide bond specificities were retained, and the activities were generally unaffected or moderately reduced at 22 degrees C and pH 8. At 37 degrees C the activity of chymotrypsin, endoproteinase Arg-C, V8 protease at pH 4, and pepsin was substantially reduced and decreased in the order methanol, ethanol, 2-propanol, and acetonitrile. The activity of thermolysin at 55 degrees C was reduced very little in the presence of 20% organic solvent and 50 mM Ca2+. In low calcium and 20% 2-propanol at 22 degrees C the activity of thermolysin was restricted to the complete and specific cleavage of peptide bonds N-terminally of Phe, Ile, and Leu. The experiments suggest that secondary proteolytic digestions can be carried out directly in reversed-phase-HPLC fractions, and that organic cosolvents can be applied to control the degree of proteolysis. Moreover, the denaturing potential of these solvents might be useful in the degradation of proteins resistant to proteolysis, for example, in studies aimed at identification of disulfide bridges.
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PMID:Generation of peptides suitable for sequence analysis by proteolytic cleavage in reversed-phase high-performance liquid chromatography solvents. 306 54

A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up.
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PMID:A novel chromatographic procedure for purification of bacterial plasmids. 631 36

The Milli-Q PF Plus water polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC) treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver, kidney, and bladder as well as cultured human corpus cavernosum smooth muscle cells (HCCSMC). RNA was prepared by solubilization in guanidinium isothiocyanate, phenol/chloroform extraction, and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (approximately 7.6kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water or Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water or Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute to DEPC-treated water for the preparation of RNA and Northern blot analysis.
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PMID:Comparison of Milli-Q PF plus water with DEPC-treated water in the preparation and analysis of RNA. 864 47

The Milli-Q PF Plus water-polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC)-treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue-cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver kidney and bladder as well as cultured human corpus cavernosum smooth muscle cells. RNA was prepared by guanidinium isothiocyanate solubilization, phenol/chloroform extraction and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (ca. 7.6 kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2 kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water for Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water and Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute for DEPC-treated water for the preparation of RNA and Northern blot analysis.
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PMID:Comparison of Milli-Q PF Plus water to DEPC-treated water in the preparation and analysis of RNA. 877 61

The complex between the ribonuclease domain of the ribosome-inactivating colicin E3 and its protein inhibitor, the cognate immunity Im3, has been crystallized and preliminary X-ray characterization has been performed. Single crystals of the 1:1 complex were grown from hanging-drop vapour-diffusion experiments using 2-propanol as a precipitant. The space group is P3(1)21 or P3(2)21, with unit-cell parameters a = b = 93.7, c = 76.2 A. When cryocooled, these crystals diffract to a resolution of 2.4 A. A search for suitable conventional heavy-atom derivatives was unsuccessful and so Im3 mutants containing engineered cysteine or methionine residues have been produced for mercury soaks and selenomethionine-labelling experiments, respectively.
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PMID:Crystallization of the cytotoxic domain of a ribosome-inactivating colicin in complex with its immunity protein. 1109 30

A robust ribonuclease protection assay is described here. In brief, total cellular RNA, carrier yeast transfer RNA, and 32P-labeled antisense riboprobes, (one or more designed to detect the RNA species being studied and another to detect a suitable RNA species to act as a loading control) are combined and made 0.5 M with respect to ammonium acetate. Absolute alcohol (2.5 vol) is added, and tubes are incubated at -20 degrees C for 30 min. Precipitated RNA and riboprobes are pelleted by centrifugation and, after removal of the supernatants, dissolved in a small volume of hybridization buffer. After hybridization for 16 h at 55 degrees C, a ribonuclease cocktail is added to digest the unhybridized RNA. This is followed by a proteinase K digestion step that degrades the ribonucleases. Finally, the hybridized complex is precipitated at -20 degrees C using isopropanol:4 M guanidium thiocyanate (2:1), with added glycogen as a coprecipitant, and harvested by centrifugation. The pellet is dissolved in loading buffer, and the sample is electrophoresed in a polyacrylamide gel. The intensities of the bands in the gel representing the protected fragments for the target RNA and the loading control are quantitated by phosphorimager analysis.
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PMID:Quantitation of RNA by ribonuclease protection assay. 1649 11

Homologous tissues, such as adipose tissue, may be an interesting source of acellular scaffolds, maintaining a complex physiological three-dimensional (3D) structure, to be recellularized with autologous cells. The aim of the present work is to evaluate the possibility of obtaining homologous acellular scaffolds from decellularization of the omentum, which is known to have a complex vascular network. Adult rat and human omenta were treated with an adapted decellularization protocol involving mechanical rupture (freeze-thaw cycles), enzymatic digestion (trypsin, lipase, deoxyribonuclease, ribonuclease) and lipid extraction (2-propanol). Histological staining confirmed the effectiveness of decellularization, resulting in cell-free scaffolds with no residual cells in the matrix. The complex 3D networks of collagen (azan-Mallory), elastic fibers (Van Gieson), reticular fibers and glycosaminoglycans (PAS) were maintained, whereas Oil Red and Sudan stains showed the loss of lipids in the decellularized tissue. The vascular structures in the tissue were still visible, with preservation of collagen and elastic wall components and loss of endothelial (anti-CD31 and -CD34 immunohistochemistry) and smooth muscle (anti-alpha smooth muscle actin) cells. Fat-rich and well vascularized omental tissue may be decellularized to obtain complex 3D scaffolds preserving tissue architecture potentially suitable for recellularization. Further analyses are necessary to verify the possibility of recolonization of the scaffold by adipose-derived stem cells in vitro and then in vivo after re-implantation, as already known for homologus implants in regenerative processes.
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PMID:Decellularized omentum as novel biologic scaffold for reconstructive surgery and regenerative medicine. 2354 63


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