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)

Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex.
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PMID:The effect of Raney nickel on the covalent thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate complex. 125 51

The addition of cationic proteins such as lysozyme, ribonuclease and cytochrome C enhanced the beta-lactam-induced bacteriolysis of staphylococci measured as release of wall label or by optical density. The treatment of staphylococci with penicillin plus cytochrome C resulted in a reduced viability of bacteria compared with those treated with penicillin alone. The wall autolysis and the penicillin-induced bacteriolysis of staphylococci were enhanced by the lysosomal enzyme cathepsin C. The penicillin-induced bacteriolysis was also enhanced by the D-amino acids D-alanine and D-methionine, while the comparable L-amino acids did not reveal any activity. On the other hand, some polyanionic substances were able to suppress the penicillin-induced bacteriolysis. Radiochemical and electron microscopic studies revealed the participation of bacterial wall autolysins in the first steps of degradation processes of staphylococcal walls within murine bone marrow-derived macrophages.
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PMID:The modulation of the bacteriolytic effect of beta-lactam antibiotics by non-antibiotics. 129 43

Two overlapping genomic clones for a rat nucleoside diphosphate kinase (NDP kinase) have been isolated and characterized. Complete sequencing of the genomic segment including the whole coding region for the enzyme revealed that the gene consists of four exons spanning 5.5 kilobase pairs. Primer extension analyses and ribonuclease protection assays indicated that the transcription may start from multiple sites with the major initiation site at 3 base pairs upstream from the translation initiation site, Met-1. Neither CAAT-box nor TATA-box could be assigned for each transcription initiation site, whereas five putative Sp1-binding sites (GC-boxes) were present in the 5'-flanking region. These features of the NDP kinase gene represent those of housekeeping genes. In genomic Southern blotting using a full-length rat NDP kinase cDNA as a probe, many positively hybridized fragments were detected. In support of this, five possible processed pseudogenes were identified in different DNA segments although many other NDP kinase-related genomic fragments remained to be characterized. These results demonstrate that the NDP kinase gene may consist of a multiple gene family.
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PMID:Isolation and characterization of a gene encoding rat nucleoside diphosphate kinase. 132 Nov 45

Hydrophobic interactions between the S-peptide and S-protein in the ribonuclease-S complex are probed using molecular dynamics simulations and free energy calculations. Three successive mutations at the buried position Met13 are simulated: Met----Leu, Leu----Ile, and Ile----Val, for which X-ray structures and experimental thermodynamic data are available. The calculations give theoretical estimates of the changes in binding free energies associated with these mutations. The calculated free energy differences are small (0-1.6 kcal/mol), in agreement with experiment. However the simulated structures deviate significantly from the experimental ones (mean deviation approximately 1.5-2 A), and a large uncertainty in the calculated free energies (1-2 kcal/mol) arises from the multiple minimum problem. Indeed, multiple conformations are available to the side chains around the mutation site, and the sampling of dihedral rotamer transitions is limited, despite long simulations. Fluctuations within each local minimum give rise to a small statistical error. However the uncertainty due to multiple conformations is much greater than the uncertainty due to random statistical errors. In our work, an artificial cancellation of errors arose because we studied conformations of the RNase complex and of the S-peptide that were very similar. In general, the criterion for a precise simulation is not merely to reduce the random statistical error, as has been suggested, but rather to sample all the important local minima along the mutation pathway, and to reduce the statistical error for each one. Our calculations suggest that the packing changes associated with the mutations are energetically small and localized, and largely cancel when the complex and the S-peptide are compared. Solvation of the methionine side chain partial charges in the S-peptide and the complex appear to be energetically equivalent, so that removing them (as in Met13----Leu, Ile, Val) does not affect binding. Enthalpy and entropy changes could not be estimated reliably.
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PMID:Thermodynamics of protein-peptide interactions in the ribonuclease-S system studied by molecular dynamics and free energy calculations. 139 Jun 51

Seven hydrophobic residues ranging in size from glycine to phenylalanine have been substituted for the wild-type methionine residue at position 13 in a 15-residue truncated version (S15) of S-peptide, the small component of ribonuclease S. Complexes of both S-15 and the seven variants with S-protein yielded isomorphous crystals. The structures of all eight complexes have been refined to final R-factors in the range of 17-19%. [See Kim, E. E. Varadarajan, R., Wyckoff, H. W., and Richards, F. M. (1992) Biochemistry (preceding paper in this issue) for the description of the reference S-15 complex.] Multiple side-chain conformations were seen for six residues in all of the complexes and for two to three additional residues in at least some of the complexes. Three of the complexes, Gly, Ala, and alpha-amino-n-butyric acid (ANB), contained a single water molecule in the cavity near residue 13 that makes three hydrogen bonds to protein atoms. Although space is available, no evidence for additional water in this region, ordered or disordered, was found. The atoms in the cavity wall tend to shrink the cavity by moving in on the small residues and to swell the cavity by moving out for the larger Phe substitution. A swelling seen with leucine was attributed to a shape effect since Leu, Ile, and Met all have the same volume. A slight volume contraction of the collection of interior residues outside of the region of position 13 was also noted. (All changes noted are in the direction to maintain a constant packing density averaged over the whole protein.) Leu51, a surface hydrophobic residue, moved considerably in the G, A, and ANB complexes in directionswhich would tend to decrease the cavity volume. The only other major change in position, 1.5 A, was the 66-69 loop, which is about 25 A from position 13. His12, Phe120, and Asp121 appear to be involved in this movement, but the connection with position 13 is not clear at all. The thermodynamic data on the association reaction for all of these complexes have been previously reported [Connelly, P. R., Varadarajan, R., Sturtevant, J. M., & Richards, F. M. (1990) Biochemistry 29, 6108-6114; Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426]. Some comments are offered on our initial attempts to correlate the structural changes with the changes in the thermodynamic parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. 146 20

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.
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PMID:Heat capacity changes for protein-peptide interactions in the ribonuclease S system. 173 99

The capacity of some Escherichia coli (E. coli) ribosomal proteins to bind to tRNA and to hydrolyse their aminoacylated derivatives has been analysed. The following results were obtained: (1) The basic proteins L2, L16 and L33 and S20 bound f[3H]Met-tRNA to a similar extent as the total proteins from 30 S (TP30) or 50 S (TP50) when tested by nitrocellulose filtration, in contrast to the more acidic proteins L7/L12 and S8. (2) The proteins of the peptidyltransferase centre, L2 and L16, showed no distinct specificity, binding various charged tRNAs from E. coli and Saccharomyces cerevisiae (S. cerevisiae). (3) A number of isolated ribosomal proteins hydrolysed aminoacyl-tRNA as assessed by trichloroacetic acid precipitation, in contrast to the TP30 and TP50. (4) The loss of radiolabel from Ac[14C]Phe-tRNA and from [14C]tRNA in the presence of these proteins could not be prevented by RNasin, a ribonuclease inhibitor, whereas that mediated by a sample of non-RNase-free bovine serum albumin was inhibited. (5) When double-labelled, Ac[3H]Phe-[14C]tRNA was incubated with L2 both radiolabels were lost, indicating that this potential candidate for a peptidyltransferase enzyme does not specifically cleave the ester bond between the aminoacyl residue and the tRNA.
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PMID:The complex between ribosomal proteins and aminoacyl-tRNA: the interactions and hydrolytic activities are not confined to the proteins L2 and L16 of Escherichia coli ribosomes. 218 27

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. Residue 13 in the peptide is methionine. According to the X-ray structure of the complex of S-protein and S-peptide (1-20), this residue is almost fully buried. We have substituted Met-13 with seven other hydrophobic residues ranging in size from glycine to phenylalanine and have determined the thermodynamic parameters associated with the binding of these analogues to S-protein by titration calorimetry at 25 degrees C. These data should provide useful quantitative information for evaluating the contribution of hydrophobic interactions in the stabilization of protein structures.
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PMID:Thermodynamics of protein-peptide interactions in the ribonuclease S system studied by titration calorimetry. 238 73

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.
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PMID:Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli. 242 55

We report studies in vitro of the interaction between non-formylated initiator Met-tRNA(fMet) and 70S ribosomes. The binding of Met-tRNA(fMet) to ribosomes carrying fMet-tRNA(fMet) in the P-site is strongly stimulated by elongation factor EF-Tu:GTP in the presence of (AUG)3. The enzymatically bound Met-tRNA(fMet) does not react with puromycin. The bound Met-tRNA(fMet) can accept formylmethionine from P-site-bound fMet-tRNA(fMet). These results demonstrate a functionally active binding at the ribosomal A-site. Partial ribonuclease digestion (footprinting) was used to study the sites in Met-tRNA(fMet) which are involved in the interaction with the ribosomal A-site. The results show that a large part of the tRNA molecule is protected by the ribosome against ribonuclease digestion. In addition to the protection found in the amino acid region and the anticodon arm, protection is seen in the D-loop and in the extra arm. No region within the bound tRNA is found to be more accessible for RNases than in the free Met-tRNA(fMet). The reported enhancement of ribonuclease cuts in the D- and T-arms of A-site-bound Phe-tRNAPhe is thus not found in A-site bound Met-tRNA(fMet).
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PMID:Interaction between non-formylated initiator Met-tRNA(fMet) and the ribosomal A-site from Escherichia coli. 244 56


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