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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lovett, James S. (Purdue University, West Lafayette, Ind.). Chemical and physical characterization of "nuclear caps" isolated from Blastocladiella zoospores. J. Bacteriol. 85:1235-1246. 1963.-Electron micrographs of Blastocladiella zoospores have shown the nuclear cap to contain essentially all of the small (250 to 300 A) electron-dense particles of the cell. Preparations of clean, whole nuclear caps were isolated to study the composition of the intact organelles and their particulate contents. The cap is strongly basophilic, and is composed of 60% protein and 40% ribonucleic acid (RNA). It represents 18% of the dry weight, and contains 69% of the total RNA, of the spore. The amino acid composition of cap proteins is similar to the ribosomal protein of other organisms. The nuclear cap contents have been extracted and isolated by high-speed centrifugation. More than 95% of the material has a sedimentation coefficient of 83S in 0.005 m Mg. The 83S particles form aggregates at higher Mg concentrations and dissociate to yield 63S and 41S peaks at low Mg concentrations. Purified cap particles contain 37% protein and 63% RNA. The RNA has a nucleotide composition (in moles per cent) of 18.5% cytidylic, 26.2% adenylic, 31.8% guanylic, and 23.5% uridylic acid. The particles contain a latent ribonuclease, which can be activated by urea, and are susceptible to degradation by added pancreatic ribonuclease. The available evidence supports a concept of the zoospore nuclear cap as an unusual intracellular "packet" of ribosomes.
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PMID:CHEMICAL AND PHYSICAL CHARACTERIZATION OF "NUCLEAR CAPS" ISOLATED FROM BLASTOCLADIELLA ZOOSPORES. 1404 13

The effects of various neutral salts on the temperature of the thermally-induced denaturation of the globular protein ribonuclease are described and compared with the effects of these salts on helix-coil transition temperatures in other macromolecules. These agents affect the stability of the native form of macromolecules as diverse as ribonuclease, collagen, DNA, and myosin in very similar ways; salts such as KSCN and CaCl(2) serve as very potent general structural destabilizers or denaturants, while salts such as (NH(4))(2)SO(4) and K(2)HPO(4) strongly stabilize the native conformation. The effectiveness of the neutral salts as ribonuclease destabilizers is compared with that of urea and the guanidinium salts.
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PMID:NEUTRAL SALTS: THE GENERALITY OF THEIR EFFECTS ON THE STABILITY OF MACROMOLECULAR CONFORMATIONS. 1416 81

Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA(ile) from the bacterium Azoarcus was probed by ribonuclease T(1) and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3'-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg(2+) and the concentration of urea, and RNase T(1) experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5' and 3' ends of unspliced pre-tRNA.
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PMID:Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA. 1512 19

Barnase, a well-characterized ribonuclease, has been decomposed into six modules (M1-M6) or secondary structure units (S1-S6). We have studied the foldability and activity of the barnase mutants obtained by permutation of the four internal modules (M2-M5) or secondary structure units (S2-S5) to investigate whether permutation of these building blocks is a useful way to create foldable and/or functional proteins. In this study, we found that one of the secondary structure unit mutants was expressed in Escherichia coli only when His102 was substituted by alanine, which is a catalytic residue of wild-type barnase. This mutant (S2354H102A) had ordered conformations, which unfolded cooperatively during urea-induced unfolding experiments. S2354H102A interacted with other barnase mutants to show a distinct RNase activity, although its own activity was quite weak. This interaction was specific, because S2354H102A interacted with only barnase mutants having His 102 and certain orders of the secondary structure units giving a distinct RNase activity. These results suggest that secondary structure units permuted in barnase mutants maintain their intrinsic "interacting ability" that is used for the folding of wild-type barnase, and the units can form certain conformations that complement those of the appropriate counterparts. Seven of 23 secondary structure unit mutants and only 2 of 23 module mutants had RNase activity. On the basis of the results of analyses of foldability and RNase activity of the mutants performed in this and previous studies, we conclude that secondary structure units are more suitable than modules as building blocks to create novel foldable and/or functional proteins in the case of barnase.
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PMID:Foldability, enzymatic activity, and interacting ability of barnase mutants obtained by permutation of secondary structure units. 1517 Mar 34

The reaction of hydroxyl and other oxygen-based radicals with the side chains of proteins on millisecond timescales has been used to probe the structure of proteins, their dynamics in solution and interactions with other macromolecules. Radicals are generated in high flux within microseconds from synchrotron radiation and discharge sources and react with proteins on timescales that are less than those often attributed to structural reorganisation and folding. The oxygen-based radicals generated in aqueous solution react with proteins to effect limited oxidation at specific amino acids throughout the sequence of the protein. The extent of oxidation at these residue markers is highly influenced by the accessibility of the reaction site to the bulk solvent. The extent of oxidation allows protection levels to be measured based on the degree to which a reaction occurs. A map of a protein's three-dimensional structure is subsequently assembled as in a footprinting experiment. Protein solutions that contain various concentrations of substrates that either promote or disrupt structural transitions can be investigated to facilitate site-specific equilibrium and time-resolved studies of protein folding. The radical-based strategies can also be employed in the study of protein-protein interactions to provide a new avenue for investigating protein complexes and assemblies with high structural resolution. The urea-induced unfolding of apomyoglobin, and the binding domains within the ribonuclease S and calmodulin-melittin protein-peptide complexes are presented to illustrate the approach.
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PMID:Photochemical and electrophysical production of radicals on millisecond timescales to probe the structure, dynamics and interactions of proteins. 1529 29

Earlier studies have reported that trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0-8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK(a) of 4.66 +/- 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-alpha-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK(a) of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK(a), whereas it stabilizes proteins at pH values above its pK(a). This conclusion was reached by determining the T(m) (midpoint of denaturation), delta H(m) (denaturational enthalpy change at T(m)), delta C(p) (constant pressure heat capacity change), and delta G(D) degrees (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T(m) and delta G(D) degrees depend on TMAO concentration at each pH value and that delta H(m) and the delta C(p) are not significantly changed in presence of TMAO.
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PMID:Counteracting osmolyte trimethylamine N-oxide destabilizes proteins at pH below its pKa. Measurements of thermodynamic parameters of proteins in the presence and absence of trimethylamine N-oxide. 1565 73

When C(14) carboxyl indoleacetic acid (IAA) is transported through Avena coleoptile sections a fraction of the activity becomes bound. The nature of this bound IAA has been investigated. Upon extraction with solvents and chromatography a substance having the R(F) of IAA in 4 solvents was detected. No evidence could be found for the formation of indoleacetyl conjugates. In pea stem sections subjected to a similar experimental regime good evidence was obtained for the occurrence of conjugates. When IAA was supplied exogenously to coleoptile sections floating in solutions the occurrence of conjugates was shown to be dependent on the presence of the primary leaf. In its absence no conjugates could be detected.On grinding coleoptile sections and subsequent centrifugation at 240 x g the radioactivity was found to be in the tissue fraction as opposed to the supernatant. The radioactivity cannot be removed from the tissue by extraction with water, buffer solution or treatment with ribonuclease. It is readily removed by 10% urea, crystalline trypsin and chymotrypsin. It is therefore concluded that IAA becomes bound to a protein. Bound IAA does not appear to be able to cause growth in Avena coleoptile sections.
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PMID:Bound indoleacetic Acid in Avena coleoptiles. 1665 59

The distribution of incorporated synthetic cytokinins (N(6)-[8-(14)C]benzyladenine ([8-(14)C]bzl(6)Ade) and N(6)[8-(14)C]furfuryladenine ([8-(14)C]fr(6)Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-(14)C]bzl(6)Ade or [8-(14)C]fr(6)Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-(14)C]bzl(6)A or [8-(14)C]fr(6)A in the rRNA peak after the fractionations. [8-(14)C]fr(6)A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.The distribution of [8-(14)C]fr(6)A moieties in mono- and oligonucleotides derived from combined treatment of [8-(14)C]fr(6)Ade-labeled rRNA with ribonuclease T(1) and pancreatic ribonuclease A was measured by fractionating the digest on a DEAE-cellulose column in the presence of 7 m urea and determining the [8-(14)C]fr(6)A content in each fraction. The [8-(14)C]fr(6)A content in the oligonucleotides varied from 46 to 210 mumol/mol of adenosine (A). The mol ratio of [8-(14)C]fr(6)A to A was three to four times greater for oligonucleotides containing uridine or cytidine ([A](n)Up or [A](n)Cp) than for those containing quanosine ([A](n)Gp).
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PMID:Distribution of incorporated, synthetic cytokinins in ribosomal RNA preparations from tobacco callus. 1666 Feb 83

This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.
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PMID:Peptide sequence and conformation strongly influence tryptophan fluorescence. 1806 77

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.
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PMID:A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea). 1820 12


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