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)

Hydrolysis of serum albumin by proteinase K was strongly (greater than 7-fold) stimulated by urea and dodecylsulfate in a dose-dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates. Although dodecylsulfate inhibited ribonuclease activity in the test-tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K. The combined action of the detergent and proteinase K was also effective in degrading "masked" proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.
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PMID:Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of 'masked' proteins. 123 99

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.
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PMID:The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes. 128 Sep 19

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.
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PMID:Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI. 131 95

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

1. A ribonuclease isolated from porcine thyroid cytosol using phenol: sodium dodecylsulfate treatment was associated with RNA and identical to latent alkaline ribonuclease. 2. Distribution of activity between aqueous and phenolic phases depended on pH, RNA, and ribonuclease inhibitor. 3. The ribonuclease was totally resistant to urea, guanidinium: HCl, chloroform:isoamyl alcohol, ethanol, heating at 100 degrees C for 10 min or at 80 degrees C plus 100 mM NaCl. It was highly resistant to hydrolysis by proteinase K except in the presence of detergent. 4. The extreme stability and other properties of latent alkaline ribonuclease could be the result of its association with RNA.
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PMID:Porcine thyroid cytosolic, latent alkaline ribonuclease: resistance to protein denaturants. 149 76

We have used equilibrium binding analyses to evaluate the influence of temperature and urea on the affinity of hen egg white lysozyme and bovine pancreatic ribonuclease A for surface-immobilized Cu(II) ions. Linear Scatchard plots suggested that these model proteins were interacting with immobilized metal ions via a single class of intermediate-affinity (Kd = 10-40 microM) binding sites. Alterations in temperature had little or no effect on the immobilized Cu(II) binding capacity of either protein. Temperature effects on the interaction affinity, however, were protein-dependent and varied considerably. The affinity of lysozyme for immobilized Cu(II) ions was significantly decreased with increased temperature (0 degree C-37 degrees C), yet the affinity of ribonuclease did not vary measurably over the same temperature range. The van 't Hoff plot (1n K vs 1/T) for lysozyme suggests a straight line relationship (single mechanism) with a delta H of approximately -5.5 kcal/mol. Urea effects also varied in a protein-dependent manner. A 10-fold reduction in the affinity of lysozyme for the immobilized Cu(II) was observed with the urea concentrations up to 3 M; yet urea had no effect on the affinity of ribonuclease for the immobilized metal ions. Although the interaction capacity of lysozyme with the immobilized Cu(II) ions was decreased by 50% in 3 M urea, ribonuclease interaction capacity was not diminished in urea. Thus, temperature- and urea-dependent alterations in protein-metal ion interactions were observed for lysozyme but not ribonuclease A. The complete, yet reversible, inhibition of lysozyme- and ribonuclease-metal ion interactions by carboxyethylation with low concentrations of diethylpyrocarbonate provided direct evidence of histidyl involvement. The differential response of these proteins to the effects of temperature and urea was, therefore, interpreted based on calculated solvent-accessibilities and surface distributions of His residues, individual His residue pKa values, and specific features of the protein surface structure in the immediate environment of the surface-exposed histidyl residues. Possible interaction mechanisms involved in protein recognition of macromolecular surface-immobilized metal ions are presented.
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PMID:Protein interactions with surface-immobilized metal ions: structure-dependent variations in affinity and binding capacity with temperature and urea concentration. 185 19

The anti-cancer drug cis-diamminedichloroplatinum (II) (cis-DDP) reacted with Tetrahymena self-splicing rRNA ribozyme, causing loss of self-splicing activity and formation of a number of platinated RNA species. The formation of one distinct platinated product, migrating at an apparent size of 2400 nt, was closely associated with ribozyme inactivation. This platinated RNA was resistant to T1 ribonuclease digestion, suggesting the presence of inter-strand Pt cross-links. The reaction rate of cis-DDP with the ribozyme followed first order kinetics and showed a saturation effect with increasing cis-DDP concentration, characteristic of an affinity-label type of interaction rather than bimolecular collision. The apparent KI for binding of cis-DDP to the ribozyme was 62 microM. Ribozyme treated with urea was not inactivated by cis-DDP, indicating that the native structure of the RNA is required for reaction with cis-DDP. Mg++, which binds to the ribozyme and causes conformational changes in the molecule, protected the ribozyme from inactivation by cis-DDP and also prevented the formation of platinated RNA. These results suggest that binding of cis-DDP to sites formed by certain secondary or tertiary structural elements of the RNA enhance the rate and the specificity of reaction of the reagent with the ribozyme.
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PMID:Inactivation of Tetrahymena rRNA self-splicing by cis-platin proceeds through dissociable complexes. 190 1

The significance of free alkaline ribonuclease (RNase) activity as a criterion of protein metabolism and nutrition in traumatized man is evaluated in this report. Plasma and urinary levels of RNase were measured in severely injured, hypermetabolic patients and in normal controls. Significant increases in the plasma and urinary RNase levels were seen in these polytrauma victims and they were positively correlated. Plasma RNase levels were also significantly related to blood urea nitrogen and daily urinary nitrogen excretion. Urinary clearance of RNase was increased by 220% in trauma victims, although the creatinine clearance was not affected by trauma. In a subgroup of eight patients who were fed intravenously (1.4 times basal energy expenditure calories and 250-300 mg of N per kilogram per day) for 6 days, the daily excretions of urinary RNase, nitrogen, 3-methylhistidine, creatinine, and catecholamines were measured. There was a significant negative correlation between daily urine RNase and nitrogen balance. A general increase in all the metabolic parameters on the first day of feeding was seen, suggesting a nutritional stress superimposed on the trauma-induced metabolic stress. Excretion of RNase, 3-methylhistidine, and creatinine peaked on the first day of feeding and then decreased. The normal levels could not be reached even after 6 days of adequate nutrition. The results suggest that RNase levels could be used as a biomarker of protein metabolism.
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PMID:Nutritional influence on the plasma and urine-free alkaline ribonuclease levels in severe trauma victims. 190 73

The two sequences that define the self-cleaving elements from the genomic and antigenomic RNA of hepatitis delta virus were folded into secondary structures with similar features. Evidence in support of the two models was obtained from limited ribonuclease digestion of genomic and antigenomic RNA fragments containing the sequence 3' of the cleavage site. Under conditions where the rates of self-cleavage are enhanced by addition of 5 M urea (2-10 mM Mg2+ at 37 degrees C), ribonucleases T1, U2, A and V1 generated digestion patterns consistent with the proposed RNA structures. The evidence for a relatively stable structure in urea when Mg2+ is present suggests that denaturant-enhanced rates of self-cleavage could result from destabilization of competing inactive structures.
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PMID:Evidence that genomic and antigenomic RNA self-cleaving elements from hepatitis delta virus have similar secondary structures. 192 26

Phosphate is a competitive inhibitor of transesterification of GpC by the ribonuclease barnase. Barnase is significantly stabilized in the presence of phosphate against urea denaturation. The data are consistent with the existence of a single phosphate binding site in barnase with a dissociation constant, Kd, of 1.3 mM. The 2D 1H NMR spectrum of wild-type barnase with bound phosphate is assigned. Changes in chemical shifts and NOEs for wild type with bound phosphate compared with free wild type indicate that phosphate binds in the active site and that only small conformational changes occur on binding. Site-directed mutagenesis of the active site residues His-102, Lys-27, and Arg-87 to Ala increases the magnitude of Kd for phosphate by more than 20-fold. The 2D 1H NMR spectra of the mutants His-102----Ala, Lys-27----Ala, and Arg-87----Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102----Ala and Lys-27----Ala have essentially the same structure as weild type, while some structural changes occur in Arg-87----Ala. It appears that phosphate binding by barnase is effected mainly by positively charge residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.
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PMID:Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR. 195 71


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