Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.4 (ribonuclease)
 6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship of structure to function in the recognition of ribonuclease S-peptide by S-protein was studied by several methods. Liquid phase peptide synthesis was employed to generate analogs of S-peptide in which from 1 to 8 residues were deleted from the NH2-terminal end of the S-peptide. Additional derivatives were made by substitutions in the NH2-terminal three amino acids or by modifying the S-peptide analogs by trifluoroacetylation. The analogs were generated in the following way. S-Peptide was cleaved with chymotrypsin. The fragment obtained, RNase(9-20), was purified and lengthened step by step using liquid phase peptide synthesis. A second set of analogs were prepared by cleavage of CF3CO-S-peptide with elastase and the resulting CF3CO-RNase(7-20), similarly lengthened. The various analogs of S-peptide were tested in their capacity to combine with S-protein and regenerate biological activity as measured by Vmax and Kb. This work shows a positive contribution of every one of the first 8 NH2-terminal residues of S-peptide to the molecular recognition of S-protein in the presence of RNA substrate. Substitution of the first 3 residues by alanine or blocking of the free amino groups decreases recognition, indicating that the original primary structure is the most favorable one.
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PMID:Ribonuclease S-peptide. A model for molecular recognition. 125 70

In an attempt to throw further light on the relationship of R17 phage RNA and A-protein during the early stages of infection, studies were carried out to determine the effect of ribonuclease (ribonuclease I, EC 3.1.4.22) on the ability of these two phage components to penetrate into host bacteria. It was found that the penetration of phage RNA is affected by ribonuclease concentrations as low as 0.1 mug/ml, while the penetration of phage A-protein was unaffected by ribonuclease concentrations as high as 20 mug/ml. In addition, it was found that a significant fraction of the phage RNA is resistant to the ribonuclease effect. This RNase-resistant portion of the phage population increased with increasing phage concentrations, and gave rise to the penetration of intact, 28S RNA molecules that produced the expected number of infectious centers. These findings are discussed in terms of a model for phage RNA injection in which the A-protein functions both as an attachment organelle and a pilot protein that guides the RNA from the capsid to the exterior surface of the F pilus, and thence into the host bacterium.
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PMID:The effect of ribonuclease on the penetration of R17 phage A-protein and RNA. 127 4

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner.
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PMID:Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart. 132 37

Two-dimensional (2D) 1H NMR experiments using deuterium labeling have been carried out to investigate the solution of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), which consists of 155 amino acids. To simplify the 1H NMR spectra, two fully deuterated enzymes bearing several protonated amino acids were prepared from an RNase HI overproducing strain of E. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, Ile, Val, and Leu. The other was labeled by only protonated His and Ile. The 2D 1H NMR spectra of these deuterated RNase HI proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.
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PMID:1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids. 133 Jan 30

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.
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PMID:A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes. 137 37

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.
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PMID:RNA abundance measured by a lysate RNase protection assay. 138 Nov 96

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

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.
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PMID:Inactivation of ribonuclease inhibitor by thiol-disulfide exchange. 144 7

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S3 and S6 proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.
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PMID:RNase X2, a pistil-specific ribonuclease from Petunia inflata, shares sequence similarity with solanaceous S proteins. 146 46

We have developed a modified RNase protection assay in which the antisense RNA probe is prepared from a PCR-amplified DNA template rather than from a linearized plasmid DNA template. In this assay, an RNA polymerase promoter sequence is attached to the 5' end of the antisense PCR primer. Using this modified antisense primer in conjunction with the paired sense primer, PCR amplification generates a linear DNA template that includes an RNA polymerase promoter sequence. Transcription in vitro initiated by the incorporated promoter in the presence of RNA polymerase and ribonucleotide triphosphates produces a radiolabeled run-off antisense RNA transcript, which can then be used as probe for RNase protection analysis. Probes generated by this method obviate the need to subclone DNA sequences into transcription vectors for synthesis of antisense transcripts. Due to the simplicity of its design and the lack of need for subcloning, this strategy offers greater flexibility than conventional methods for the production of single-stranded RNA probes, and thus facilitates the implementation of the ribonuclease protection assay.
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PMID:Application of the polymerase chain reaction to the ribonuclease protection assay. 147 48


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