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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The mechanism of the reaction between ribonuclease and GSH at elevated temperatures has been studied by using N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide to label the reduced ribonuclease. 2. After incubation for 2hr. at 35 degrees , enzymically active ribonuclease was recovered; at 50.8 degrees half of the initial ribonuclease was recovered as enzymically active ribonuclease and half as reduced labelled ribonuclease; at 55 degrees all of the initial ribonuclease was recovered in the labelled form. 3. It was inferred that the rate-limiting step was the reduction of the first disulphide bond in any one molecule. This was followed by rapid reduction of the other bonds in the same molecule.
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PMID:Reduction of ribonuclease by glutathione at elevated temperatures: the molecular mechanism. 604 89

Reduced RNase A was reoxidized, and the incorrectly formed disulfide bonds were reshuffled to the native ones by oxidized and reduced glutathiones, as described in the first paper of this series. The intermediates in the regeneration of the disulfide bonds were trapped without any chemical modification and were fractionated on a carboxymethylcellulose column at pH 3.5 with a salt gradient. The elution curves of the partially regenerated RNase A from the carboxymethylcellulose column were obtained by measurement of the absorption at 275 nm and by determination of the SH content (of cysteine residues) and consisted of 11 fractions, G8, G7, G6, G5, G4, G3, G2, G1, G0, N, and F. Some of the fractions were isolated, and their measured molecular weights were consistent with those of monomeric RNase A. Fraction F had a molecular weight between that of the monomer and dimer, so that this fraction could not be identified. The regeneration pathway could be represented in terms of two simple reactions, RNase A(-SH) + GSSG in equilibrium or formed from RNase A(-SSG) + GSH and RNase A(-SH-SSG) in equilibrium RNase A(greater than S2) + GSH, which produced 24 monomeric intermediates (not counting the fully reduced and the native species), which differed from each other in their amino acid composition. These 24 intermediates, plus the fully reduced protein, were assigned to fractions G8--G0 (as indicated in the last column of Table I), with the aid of data from amino acid analysis, SH content, and the elution position on the carboxymethylcellulose column chromatogram. Since the regeneration reaction rapidly reached a preequilibrium among the intermediates and the fully reduced RNase A prior to the rate-limiting steps, i.e., the relative concentrations of the intermediates and fully reduced RNase A became constant with reaction time, the populations of some of the intermediates in preequilibrium were estimated by curve fitting of the elution pattern from the carboxymethylcellulose column chromatogram. The equilibrium constants among the intermediates were calculated from their populations at preequilibrium. These equilibrium constants were "extrapolated" to other intermediates whose populations could not be estimated by curve fitting, and the relative populations of all of the possible intermediates at preequilibrium were thereby represented as a function of the concentrations of reduced and oxidized glutathiones. The regeneration process was also restarted from several of the isolated intermediates, and the resulting distribution of intermediates was consistent with that from which the equilibrium constants were determined, supporting the representation of the regeneration pathways in terms of two simple reactions. Thus, the equilibrium treatment of the regeneration pathways was useful to characterize the preequilibrium state, i.e., to identify the intermediates prior to the rate-limiting steps in the pathways and to estimate their stabilities at preequilibrium at various concentrations of reduced and oxidized glutathiones.
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PMID:Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates, and equilibrium treatment. 626 76

This paper is concerned with the pathways for the regeneration of RNase A from the reduced protein by a mixture of GSSG and GSH. Experimental work on the regeneration has led to the identification of several different pathways, depending on the concentrations of GSH and GSSG, and an energetic analysis has provided information about the stabilities of the various intermediates. The equilibrium and kinetic data for the regeneration process have led to two models of protein-folding pathways. The intermediates in the regeneration process were trapped without chemical modification, and were fractionated on a carboxymethyl-cellulose column. The regeneration pathway(s) could be represented in terms of two simple reactions (Eqs. (1) and (2)). The system rapidly reaches a pre-equilibrium among the intermediates prior to the rate-limiting steps, and the concentrations of the intermediates (and hence the equilibrium constants among them) were determined. The regeneration process was also re-started from several of the isolated intermediates, and led to the predicted distribution of intermediates in the pre-equilibrium. Kinetic data, obtained by following the time dependence of the regain of enzymatic activity, together with the distributions of the intermediates at pre-equilibrium, led to the identification of the rate limiting steps, which differed according to the concentrations of GSH and GSSG. The relative apparent standard state conformational chemical potentials of the intermediates were estimated by using data for the apparent equilibrium constants (among the species in pre-equilibrium) and for the redox potentials of cysteine/cystine and GSH/GSSG. The two models deduced from the equilibrium and kinetic data are designated as growth-type and rearrangement-type models. In the growth-type model, nucleation of the native-like structure occurs in the folding process, in the rate-limiting step(s), and subsequent folding around the nucleation sites proceeds smoothly to form the native disulfide bonds and conformation. In the rearrangement-type model, proper nucleation does not occur in the folding process; instead, non-native interactions play a significant role in the folding pathways and lead to metastable intermediate species. Such non-native interactions must be disrupted or rearranged to nucleate the native interactions (in the rate limiting step(s)) for the protein to fold. Other protein foldings, reported in the literature, can be shown to conform to this model.
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PMID:Multiple pathways for regenerating ribonuclease A. 639 20

A cytosol thioltransferase was purified 37,000-fold from bovine liver by essentially the same procedure as reported for rat liver enzyme by Axelsson et al. [1978) Biochemistry 17, 2978-2984). The purified enzyme appears to be homogeneous on sodium dodecyl sulfate (SDS)-gel electrophoresis and has a molecular weight (Mr) of 11,000, an isoelectric point (pI) of 8.1, and an optimum pH with S-sulfocysteine and GSH as substrates of 8.5. It is specific for disulfides including L-cystine, S-sulfocysteine, ribonuclease A, trypsin, soybean kunitz trypsin inhibitor, soybean Bowman Birk trypsin inhibitor and insulin, and converts Bowman Birk trypsin inhibitor to an inactive form. The enzyme does not act as a protein : disulfide isomerase, as measured by reactivation of "scramble" ribonuclease and Kunitz soybean trypsin inhibitor. Thioltransferase activity was found in cytosol of various bovine tissues.
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PMID:Purification and some properties of bovine liver cytosol thioltransferase. 646 49

Refolding of dimeric porcine cytosolic or mitochondrial malate dehydrogenases and of tetrameric pig heart and skeletal muscle lactate dehydrogenases (containing 5-7 cysteine residues), as well as reformation of the four cystine cross-bridges of bovine pancreatic ribonuclease, were studied in the presence of reduced and oxidized glutathione (GSH and GSSG). At the intracellular GSH level (5 mM) reduced ribonuclease can be reoxidized by 0.01-0.5 mM GSSG (pH 7.4) both at 20 degrees C and 37 degrees C. In this physiological range of GSSG concentrations and pH, the dehydrogenases show at least partial reactivation. With GSSG concentrations greater than 5 mM, reactivation is found to be completely inhibited for all the enzymes given. The results show that at the intracellular level of GSH and GSSG, thiol groups in reduced, unfolded ribonuclease are oxidized to form intramolecular cystine cross-bridges, while thiol groups of typical cysteine enzymes, such as lactate and malate dehydrogenase, remain in their reduced state during refolding. The rate of reactivation of lactate dehydrogenase (porcine muscle) is not affected by GSSG. In the case of ribonuclease, increasing concentrations of GSSG increase the rate of reactivation: At 20 degrees C, the halftime of the correct disulfide bond formation varies from approximately equal to 80 h in the presence of 0.01 mM GSSG to approximately equal to 10 h in the presence of 0.25 mM GSSG. A further increase in the rate of reactivation at higher GSSG concentrations is accompanied by a decrease in yield. Reactivation of ribonuclease is also observed at the low glutathione level found in blood plasma (5-25 microM GSH).
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PMID:Influence of glutathione on the reactivation of enzymes containing cysteine or cystine. 661 43

Protein folding, associated with isomerization of disulfide bonds, was studied using the mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) as a stable soluble and homogeneous starting material; conditions were selected to model those within the lumen of the endoplasmic reticulum where native disulfide bonds are formed in protein biosynthesis. Folding was initiated by addition of free glutathione (GSH +/- GSSG) to promote thiol-disulfide interchange and was monitored by intrinsic protein fluorescence, appearance of native ribonuclease activity, HPLC, and nonreducing SDS-PAGE. All the analyses indicated that native RNase T1 was recovered in high yield in a variety of redox conditions. Appearance of native activity followed first-order kinetics; kinetic analysis of the intrinsic fluorescence changes indicated an additional rapid process in some conditions, interpreted as the formation of a nonnative intermediate state. Analysis by HPLC and SDS-PAGE also indicated the formation of transient intermediates. In 1.5 M NaCl, GS-RNase T1 adopts a compact native-like conformation; refolding by thiol-disulfide interchange in these conditions was accelerated approximately 2-fold. Refolding of GS-RNase T1 was catalyzed by protein disulfide isomerase (PDI); substoichiometric quantities of PDI accelerated refolding several-fold. GS-RNase T1 refolding was inhibited by BiP; refolding was completely blocked in presence of a 5-fold molar excess of BiP, and the yield of refolding was substantially reduced by equimolar concentrations of BiP; the refolding was then restored by the addition of ATP. GS-RNase T1 is a convenient model substrate for studying protein folding linked to native disulfide formation in conditions comparable to those within the lumen of the endoplasmic reticulum.
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PMID:Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate. 762 8

Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 microM reduced and denatured ribonuclease RNase-(SH)8 in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S2)4, a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 microM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'0 of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO3(2-)), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.
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PMID:Glutaredoxin accelerates glutathione-dependent folding of reduced ribonuclease A together with protein disulfide-isomerase. 771 72

Prior studies have suggested that heart expresses only the M2 isoform of the muscarinic receptor (Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.H., Ramachandran, J., and Capon, D.J. (1987) EMBO J. 6, 3923-3929). Tietje and Nathanson (Tietje, K.M., and Nathanson, N. M. (1991) J. Biol. Chem. 266, 17382-17387) have recently demonstrated that the chick heart may be unique since it expresses both the M2 and M4 isoforms of the muscarinic receptor. In this study, in order to determine whether other isoforms of the muscarinic receptor were present in the chick heart, a chick M3 muscarinic receptor receptor was cloned, characterized, and its expression in chick tissues determined. Using a human M3 muscarinic receptor cDNA as a probe, a 2.4-kilobase pair cDNA was isolated from a chick brain cDNA library which contained an open reading frame coding for a 639 amino acid protein. This protein demonstrated an 87 and 86% homology to the human and rat M3 muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were stably transfected with the chick M3 muscarinic receptor and one clone (CHO-CM3) expressed the M3 receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/- S.E., n = 3) fmol/mg protein with a Kd of 76 +/- 17 pM. This receptor demonstrated a rank order of potency for muscarinic antagonist binding characteristic for the M3 receptor: with high affinity binding for hexahydrosiladifenidol, Kd: 16 +/- 2 nM (+/- S.E., n = 3); intermediate affinity for pirenzepine, Kd: 383 +/- 47 nM, and low affinity for methoctramine, Kd: 533 +/- 185 nM (+/- S.E., n = 3). Carbamylcholine stimulation of CHO-CM3 cells resulted in a 1.6-fold increase in cyclic AMP accumulation and a 3.5-fold increase in a pertussis toxin-insensitive inositol phosphate release. These data demonstrate that the chick M3 muscarinic receptor has the properties characteristic of M3 receptors from other species. RNase protection studies demonstrated the presence of M3 muscarinic receptor mRNA in the brain, atria, and ventricle of chicks 17 days in ovo. Hence, the chick heart appears to have the unique capacity to express mRNAs coding not only for the M2 and M4 muscarinic receptors but also for the M3 muscarinic receptor.
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PMID:A novel M3 muscarinic acetylcholine receptor is expressed in chick atrium and ventricle. 792 87

The oxidative folding mechanisms of two Escherichia coli periplasmic proteins, alkaline phosphatase and RTEM-1 beta-lactamase, have been examined in vitro and in vivo. In contrast to eukaryotic proteins, which require a relatively reducing environment for optimal folding rates, both alkaline phosphatase and beta-lactamase fold fastest under very oxidizing conditions. For example, bovine pancreatic ribonuclease exhibits an optimal folding rate in a redox buffer consisting of 1 mM GSH and 0.2 mM GSSG (Lyles, M. M., and Gilbert, H. F. (1991) Biochemistry 30, 613-619); however, both E. coli alkaline phosphatase and beta-lactamase exhibit optimal in vitro folding rates at low concentrations of GSH (< 0.4 mM) and very high concentrations of GSSG (4-8 mM). For both bacterial proteins, GSH inhibits oxidative folding. Under optimal redox conditions, the rate-limiting step for the in vitro oxidative folding of alkaline phosphatase depends on the concentration of the protein, consistent with a mechanism involving rapid oxidation followed by slow dimerization. With beta-lactamase, the oxidative folding mechanism involves a competition between disulfide bond formation and folding of the molecule into a catalytically active conformation that buries the 2 reduced cysteines in the core of the enzyme. The effects of including a thiol reductant in the growth medium on the in vivo folding of alkaline phosphatase and beta-lactamase are similar to the effects observed during in vitro folding of these enzymes. The levels of both oxidized proteins are decreased by GSH in the growth medium. However, addition of a disulfide oxidant to the growth medium does not positively affect the production of either enzyme. These observations are consistent with the idea that the oxidative folding mechanisms of E. coli periplasmic proteins and, by inference, proteins of the eukaryotic endoplasmic reticulum have evolved to accommodate constraints placed on the folding reaction by the folding environment. The consequences of differences between the folding mechanisms in eukaryotic and prokaryotic disulfide-containing proteins on the expression of eukaryotic proteins in the bacterial periplasm are discussed.
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PMID:Effect of redox environment on the in vitro and in vivo folding of RTEM-1 beta-lactamase and Escherichia coli alkaline phosphatase. 796 90

Tumor cell resistance to many chemotherapeutic agents, including alkylating agents, cisplatin, and doxorubicin, is frequently associated with increased intracellular levels of the nonprotein sulfhydryl glutathione (GSH). Recent evidence has demonstrated that increased GSH levels can be accompanied by an increase in the activity of gamma-glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting step in de novo synthesis of GSH, and by an increase in the steady state level of mRNA for the catalytic subunit of GCS. Using melphalan-resistant DU 145/M4.5 human prostate carcinoma cells, which express elevated GSH levels, GCS enzyme activity, and GCS mRNA levels, we sought to determine the mechanism(s) responsible for the increased GCS mRNA expression. As determined by Northern analyses and RNase protection assays, the steady state level of GCS message in the resistant cells was increased 10-20-fold, in comparison with the drug-sensitive parent DU 145 cells. No significant difference in gene copy number or evidence of rearrangement was detected in the resistant cell line by Southern analyses. The GCS-specific mRNA isolated from the resistant cells was less stable than that isolated from the drug-sensitive cells (half-lives of 6 hr and 9 hr, respectively), indicating that this difference does not contribute to the increased steady state levels in the resistant cells. Nuclear run-on experiments revealed that the GCS transcription rate in the DU 145/M4.5 cells was increased approximately 12-fold, in comparison with that detected in the DU 145 cells. This difference in transcription rate was comparable in magnitude to the difference in steady state mRNA levels detectable in the two cell populations. Similar correlations between steady state GCS mRNA levels and transcription rates were also observed in other DU 145 lines expressing intermediate degrees of resistance to melphalan and correspondingly intermediate GCS mRNA elevations. These data suggest that GCS expression is transcriptionally regulated in these melphalan-resistant tumor cells.
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PMID:Transcriptional up-regulation of gamma-glutamylcysteine synthetase gene expression in melphalan-resistant human prostate carcinoma cells. 796 79


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