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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The degradation of S--S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S--S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S--S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S--S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S--S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S--S bonds of some other proteins. The initial product of S--S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins.
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PMID:Degradation of protein disulphide bonds in dilute alkali. 721 43

We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.
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PMID:Simple method for extracting RNA from cultured cells and tissue with guanidine salts. 768 4

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.
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PMID:Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions. 800 82

A microdissection technique for quantitation of neurochemicals in discrete brain nuclei has been applied to quantitative measurement of mRNA. The method permits quantitation of low abundance mRNA from submilligram amounts of tissue (10-500 micrograms protein). Discrete nuclei and other regions of the brain are solubilized in concentrated guanidine thiocyanate solution, mRNA is directly hybridized with riboprobes, and detected with a ribonuclease protection assay. This method eliminates the necessity for RNA isolation from solid tissue. No assumptions regarding RNA recovery are necessary since tissue specimens are solubilized, hybridized and treated with ribonuclease in a single tube. We have determined the mRNA levels of calretinin, a predominantly neuron-specific calcium binding protein in microdissected nuclei and other regions of rat brain. For interassay comparison, measurement of sample protein and beta-actin mRNA permits normalization and quantitation in terms of these internal controls. The quantity of calretinin mRNA ranged from 281 +/- 35 fg/micrograms protein in the thalamic paraventricular nucleus to 2.3 +/- 0.5 fg/micrograms protein for the cerebral cortex. The calretinin/beta-actin ratios ranged from 79.9 +/- 9.3% to 1.3 +/- 0.1%, respectively. The combination of microdissection techniques with a lysate RNase protection assay: (1) establishes this technique as quantitative for detection of high and low abundance mRNAs from microdissected brain specimens; (2) bypasses the inefficiencies and uncertainties associated with isolating RNA; and (3) enables large numbers of determinations from discrete brain nuclei to be analyzed in 2 to 3 days.
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PMID:Quantitative measurement of calretinin and beta-actin mRNA [correction of mRNAIN] in rat brain micropunches without prior isolation of RNA. 830 61

The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidine hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectroscopies. Only a single exponential process was detected for the fast time scale unfolding (rate constants from 0.014 to 0.54 s-1, depending on the final denaturant concentration). For refolding, the far-UV CD value largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase). This phase was followed by either one or two phases, with rate constants from 0.035 to 2.45 s-1 as detected by CD and fluorescence, respectively. Although this protein has a single cis-Pro residue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding. The difference in the amplitudes of the burst phases for refolding in the far- and near-UV CD spectra revealed that an intermediate state exists, with the characteristics of a molten globule. Because the one-phased fast exponential process detected by CD corresponds to the slower of the two phases detected by fluorescence, the intermediate detected by CD might be the most stable. GuHCl denaturation experiments revealed that this intermediate cooperatively unfolds, with a transition midpoint of 1.33 +/- 0.03 M. The Gibbs free energy difference (delta G) between the intermediate and the unfolded states, under physiological conditions (25 degrees C, pH 5.5, and 0 M GuHCl), was estimated to be 20.0 +/- 2.3 kJ mol-1. Therefore, it is reasonable to assume that the refolding intermediate, rather than the unfolded state, is the latent denatured state under physiological conditions. Approximately linear relationships between the GuHCl concentration and the logarithm of the microscopic rate constants determined by CD and fluorescence were also observed. By extrapolation to a GuHCl concentration of 0 M, activation Gibbs free energies of 98.5 +/- 1.1 kJ mol-1 for unfolding and 69.5 +/- 0.2 kJ mol-1 for refolding under physiological conditions were obtained. The hydrogen-exchange-refolding competition combined with two-dimensional NMR revealed that the amide protons of alpha-helix I are the most highly protected, suggesting that alpha-helix I is the initial site of protein folding. The CD and NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule.
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PMID:Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study. 852 28

The thermal stabilities of ribonuclease A (RNase A) and ribonuclease B (RNase B), which possess identical protein structures but differ by the presence of a carbohydrate chain attached to Asn34 in RNase B, were studied by proteolysis and UV spectroscopy at pH 8.0. Proteolysis was quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. Increasing protease concentrations led to a hyperbolic increase of the rate constants of proteolysis. With thermolysin, which attacks the unfolded molecules only, the thermal unfolding constants were determined by extrapolating the rate constants of proteolysis to infinite concentration of protease. With trypsin, the unfolding constants of RNase A could be confirmed. Subtilisin attacked even the native RNases, where RNase B was more stable toward proteolytic degradation. Kinetic stabilities (deltaG++) calculated from the unfolding constants for temperatures between 52.5 and 65 degrees C revealed a higher kinetic stability of RNase B, which results from enthalpic effects only, whereas entropic effects counteract stabilization. delta deltaG++ at the transition temperature of RNase A (60.4 degrees C) was 2.2 +/- 0.3 kJ mol(-1). Thermodynamic stabilities (deltaG) were estimated from the thermal transition curves at 287 nm for the temperature range from 55 to 70 degrees C. For 17.5-25 degrees C, deltaG values were determined from transition curves of unfolding induced by guanidine hydrochloride and extrapolation of the free energy values to those in the absence of denaturant. At all temperatures, RNase B proved to be more stable than RNase A with essentially the same enthalpy and entropy of unfolding. delta deltaG was 2.5 +/- 0.2 kJ mol(-1) at 60.4 degrees C and 2.3 kJ mol(-1) at 25 degrees C.
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PMID:Kinetic and thermodynamic thermal stabilities of ribonuclease A and ribonuclease B. 904 16

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.
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PMID:Protein stability: functional dependence of denaturational Gibbs energy on urea concentration. 1002 41

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding.
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PMID:Observation of multistate kinetics during the slow folding and unfolding of barstar. 1041 90

The effects of chronic treatment with haloperidol on sigma (sigma) receptors were investigated across brain regions and species. The regional distribution of [3H](+)-pentazocine binding to sigma(1) receptor was similar between the guinea pig and rat brains. The highest level of binding was detected in the brain stem and lowest in the striatum and hippocampus. The regional distribution of [3H]1, 3-di (2-tolyl) guanidine ([3H]DTG) binding in the presence of 100 nM (+)-pentazocine to sigma(2) receptor was similar to that of the [3H](+)-pentazocine binding in the guinea pig brain, while in the rat brain high levels of [3H]DTG binding were detected in the cortex, frontal cortex and cerebellum. The intraperitoneal administration of 2 mg/kg of haloperidol to guinea pig and rats once a day for 21 days produced inhibition of [3H](+)-pentazocine binding but did not affect [3H]DTG binding to sigma(2) receptors in any brain region examined. The effects of haloperidol on [3H](+)-penazocine binding in the rat were much weaker than those in the guinea pig. The regional distribution of the level of sigma(1) receptor mRNA determined by the ribonuclease protection assay was similar to that of the [3H](+)-pentazocine binding activity, except in the cortex and cerebellum where the levels of sigma(1) receptor mRNA were low in guinea pig and rat. Treatment with haloperidol did not affect the levels of sigma(1) receptor mRNA in any brain region in either species. These findings suggested that the sigma receptors differentially distributed in brain regions are down regulated by treatment with haloperidol across sigma receptor subtypes and animal species without changing the transcriptional activity of the sigma(1) receptor. The mechanisms by which sigma receptors could be differently regulated in vivo by chronic treatment with haloperidol in different species may contribute to the therapeutic efficacy of haloperidol.
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PMID:Repeated haloperidol treatment decreases sigma(1) receptor binding but does not affect its mRNA levels in the guinea pig or rat brain. 1093 88

A genetic method for isolating a mutant enzyme of ribonuclease HI (RNase HI) from Thermus thermophilus HB8 with enhanced activity at moderate temperatures was developed. T. thermophilus RNase HI has an ability to complement the RNase H-dependent temperature-sensitive (ts) growth phenotype of Escherichia coli MIC3001. However, this complementation ability was greatly reduced by replacing Asp(134), which is one of the active site residues, with His, probably due to a reduction in the catalytic activity. Random mutagenesis of the gene encoding the resultant D134H enzyme, followed by screening for second-site revertants, allowed us to isolate three single mutations (Ala(12) --> Ser, Lys(75) --> Met, and Ala(77) --> Pro) that restore the normal complementation ability to the D134H enzyme. These mutations were individually or simultaneously introduced into the wild-type enzyme, and the kinetic parameters of the resultant mutant enzymes for the hydrolysis of a DNA-RNA-DNA/DNA substrate were determined at 30 degrees C. Each mutation increased the k(cat)/K(m) value of the wild-type enzyme by 2.1-4.8-fold. The effects of the mutations on the enzymatic activity were roughly cumulative, and the combination of these three mutations increased the k(cat)/K(m) value of the wild-type enzyme by 40-fold (5.5-fold in k(cat)). Measurement of thermal stability of the mutant enzymes with circular dichroism spectroscopy in the presence of 1 M guanidine hydrochloride and 1 mM dithiothreitol showed that the T(m) value of the triple mutant enzyme, in which all three mutations were combined, was comparable to that of the wild-type enzyme (75.0 vs 77.4 degrees C). These results demonstrate that the activity of a thermophilic enzyme can be improved without a cost of protein stability.
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PMID:Enhancement of the enzymatic activity of ribonuclease HI from Thermus thermophilus HB8 with a suppressor mutation method. 1105 82


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