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)

Ultraviolet light-induced free radical alkylation with 2-propanol or D-ribose, initiated with di-tert-butyl peroxide, of poly (G), poly (U20G), and poly(A) led to the substitution of the appropriate group for the H-8 atom of the purines and addition across the 5,6-double bond of the pyrimidines. The alkylated polynucleotides were subjected to nucleolytic digestion with several nucleases. T1-RNase digestion of poly(G) irradiated with 2-propanol gave a mixture of the modified and non-modified mononucleotides. Similarly, pancreatic RNase digestion of the irradiated poly(U20G) resulted in a mixture of the appropriate mononucleotides. A T2-RNase treatment of poly(A) irradiated with 2-propanol gave the modified Ado-21:3'-P, while T2-RNase digestion of poly(A) irradiated with D-ribose led to the cyclic modified mononucleotides, in addition to the modified mononucleotides.
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PMID:Light-induced free radical alkylation of polynucleotides and their enzymatic digestion. 84 Jun 44

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 simple and cheap method of plasmid DNA preparation from both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) organism is presented here. In this method, in place of the high-priced chemicals lysostaphin and lysozyme which are commonly used for removal of cell-wall during plasmid DNA preparation from gram-positive and gram-negative bacteria, respectively, only sucrose has been used. Firstly, bacteria is treated with Trizma (pH 8.0) containing 100% sucrose (hypertonic solution). Due to this osmotic shock, protoplasm covered by the plasma membrane of bacteria possibly shrinks and becomes detached from the cell-wall. Osmotically sensitive cells thus formed, from gram-positive (S. aureus) and gram-negative (E. coli) bacteria, are finally lysed by the lysis mixture, containing brij 58 and sodium deoxycholate. The lysate is centrifuged at 15,000 rpm for 30 min to pellet the cell debris. The supernatant containing plasmid DNA is treated with either polyethylene glycol or isopropanol. The precipitate which contains plasmid DNA is dissolved in a buffer containing Tris, EDTA, NaCl, and sodium dodecyl sulfate (pH 8.0); thus protein is denatured and removed. Finally, RNA is removed by RNase treatment. The average yield of staphylococcal plasmid DNA as well as plasmid pBR322 from E. coli HB101 in 100% sucrose-treated preparations is greater than that of lysostaphin- and lysozyme-treated preparations. This method is applicable for both large-scale and small-scale preparations. The substrate activity for restriction enzyme, cloning, transforming ability, and electron microscopic profile of the plasmid DNA prepared by this method remains unaltered.
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PMID:A new method of plasmid DNA preparation by sucrose-mediated detergent lysis from Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive). 254 9

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

Study of the RNases of human body fluids has been facilitated by use of activity staining following SDS-polyacrylamide gel electrophoresis. Commercial SDS preparations contain minor lipophilic contaminants (less than 0.1%) which interfere with enzyme renaturation and prevent activity staining unless gels are washed after electrophoresis in 25% isopropanol. Partial characterization of the RNases of serum, urine, and cerebrospinal fluid (CSF) is described, including evidence that the RNases comprising bands A-C of urine and 1-3 of CSF are glycoproteins. Evidence is presented that the major RNase activities of serum (RNases 1-5) and urine (band A) do not originate in pancreas, and that leukocytes are the source of band D RNase of urine, as well as of minor RNase activities of serum and CSF. Results are summarized suggesting that elevated plasma RNase levels may be of dubious utility in the diagnosis of most malignant diseases. Some elevated levels reported in the literature may reflect the advanced age of cancer patients, negative nitrogen balance, and other secondary effects of diseases, particularly kidney dysfunction.
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PMID:Human body fluid ribonucleases: detection, interrelationships and significance. 731 42

A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper. A subequilibrium concentration of 1 microM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human- and mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously.
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PMID:TO-PRO-3 iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry. 753 Jun 20

We have developed a very efficient and rapid method for the preparation on a small or large scale of highly purified plasmid DNA from Escherichia coli. The procedure consists of five steps: (1) cell lysis by NaOH-SDS, (2) precipitation of cell lysate with 2 M potassium acetate-1 M acetic acid, (3) precipitation of the resulting supernatant with isopropanol, (4) treatment of the precipitate with RNase, and (5) a second isopropanol precipitation. The new procedure yields a plasmid DNA that is more than 90% in the supercoiled form and virtually free from proteins, RNA, and chromosomal DNA. We have thoroughly tested the method in the preparation of several thousand samples of different plasmids from various E. coli strains. We found that it consistently produced samples of plasmid DNA suitable for all routine uses such as restriction analysis, sequencing, and preparation of DNA probes for cloning and hybridization experiments. Moreover, plasmids purified by this procedure could fully replace plasmids purified on CsCl gradients for more demanding tasks such as the in vitro synthesis of RNA probes by phage RNA polymerases, the generation of deletion mutants with exonuclease III, and the transfection of mammalian cells by the calcium phosphate coprecipitation method, as tested on human fibroblasts and on CV-1 cells.
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PMID:A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli. 821 82


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