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)

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.
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PMID:Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation. 1007 77

We have shown previously that epithelial cells may contribute to the inflammatory response in the lung after exposure to crystalline silica through the production of and response to specific chemokines and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica exposure remain unclear. We hypothesize that non-oxidant-mediated silica-cell interactions lead to the upregulation of tumor necrosis factor-alpha (TNF-alpha), whereby TNF-alpha-induced generation of reactive oxygen species (ROS) leads to the activation of the monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 genes. Using a murine alveolar type II cell line, murine lung epithelial (MLE)-15, we measured the early changes in TNF-alpha, MCP-1, and MIP-2 mRNA species after exposure of the cells to 18 micrograms/cm2 silica (cristobalite) in combination with various antioxidants. Total mRNA was isolated and assayed using an RNase protection assay after 6 h of particle exposure. We found that extracellular GSH could completely attenuate the cristobalite-induced expression of MCP-1 and MIP-2 mRNAs, whereas TNF-alpha mRNA levels were unaltered. We also found using the oxidant-sensitive dye 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) that treatment of MLE-15 cells with cristobalite and TNF-alpha (1 ng/ml) resulted in ROS production. This ROS production could be inhibited with extracellular GSH treatment, and in the case of cristobalite-induced ROS, inhibition was also achieved with an anti-TNF-alpha antibody. The results support the hypothesis that TNF-alpha mediates cristobalite-induced MCP-1 and MIP-2 expression through the generation of ROS.
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PMID:Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-alpha-induced oxidant stress. 1036 23

In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.
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PMID:Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin. 1044 Nov 34

An important role of C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) in the formation of the three-dimensional structure was previously implied. In this study, we replaced the C-terminal amino acid, Val124, with amino acid residues with different properties by site-directed mutagenesis. The recombinant mutant enzymes were purified and subjected to a refolding study after being converted to a fully reduced and denatured state. There was a significant difference among the mutant enzymes in the rate of recovery of the activity when air oxidation was performed: the rate decreased in the order of V124E, V124L, V124G, V124K, V124A, and V124W. On the other hand, the recovery rates for all the mutant RNase A in the presence of GSH and GSSG were almost the same. The recovered activity of V124E after 24 h incubation reached approximately 90% of that of the wild type enzyme, followed by V124L 80%, V124A and V124W 65%, and V124K and V124G 50%. The duration of the initial lag phase became shorter in the order of V124W, V124A, V124K or V124G, V124E, or V124L. The results imply that the C-terminal amino acid significantly influences the formation of correct disulfide bonds during the refolding process and that the hydrophobic interaction of Val124 is important for efficient packing of the RNase A molecule.
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PMID:Effect of mutagenic replacement of the carboxyl terminal amino acid, val124, on the properties and regeneration of bovine pancreatic ribonuclease A. 1078 98

Gamma-glutamylcysteine synthetase (gamma-GCS) is a heterodimer consisting of heavy (gamma-GCSh) and light (gamma-GCSl) subunits. gamma-GCS catalyzes the rate-limiting de novo biosynthesis of glutathione (GSH), an abundant physiological antioxidant that plays important roles for regulating oxidative stress. Expression of gamma-GCSh and gamma-GCSl are sensitive to oxidative stress. To investigate whether expression of gamma-GCS is correlated with tumor progression, we used immunohistochemical approaches to examine 16 human colorectal adenomas and resected 57 carcinomas from untreated patients. In adjacent normal colorectal epithelium, levels of gamma-GCSh expression were low. Strong cytoplasmic staining for gamma-GCSh was detected in 3 (18.8%) adenoma and 48 (84.2%) carcinomas. The frequency of gamma-GCSh expression in carcinoma was significantly higher than in adenoma (p<0.0001). We used RNase protation assay and Western blot to determine levels of gamma-GCSh mRNA and protein from 10 pairs of matched carcinomas with adjacent normal controls. Elevated expression of both gamma-GCSh mRNA and protein were found in 6 cases, suggesting that transcriptional and/or posttranscriptional regulation play an important role in the upregulation of gamma-GCS during colorectal carcinogenesis. We also examined the expression of another redox-regulated gene, multidrug resistance protein 1 (MRP1). Strong staining for MRP1 was detected in 1 (6.3%) adenoma and 40 (70.2%) carcinomas. The frequency of MRP1 expression in carcinoma was significantly higher than in adenoma ( p<0.0001). Nuclear p53 expression was detected in 30 (52.6%) of carcinomas. There is a significant correlation between gamma-GCSh and MRP1 expression (p=0.013) but not between gamma-GCSh and p53. Since gamma-GCS is a sensor of oxidative stress, these results are consistent with the notion that oxidative stress is associated with colorectal tumor progression.
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PMID:Expression of heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) in human colorectal carcinoma. 1177 39

The C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) form a core structure in the initial stage of the folding process that leads to the formation of the tertiary structure. In this paper, roles of the C-terminal four amino acids in the structure, function, and refolding were studied by use of recombinant mutant enzymes in which these residues were deleted or replaced. Purified mutant enzymes were analyzed for their secondary structure, thermal stability, and ability to regenerate from the denatured and reduced state. The C-terminal deleted mutant enzymes showed lower hydrolytic activity for C>p and nearly identical CD spectra compared with the wild-type enzyme. The rate of recovery of activity was significantly different among the C-terminal deleted mutant enzymes when air oxidation was employed in the absence of GSH and GSSG: the rates decreased in the order of des-124-, des-(123-124)-, and des-(122-124)-RNase A. It is noteworthy that the regeneration rates of mutant RNase A in the presence of GSH and GSSG were nearly the same. Des-(121-124)-RNase A failed to recover activity both in the presence and absence of glutathione, due to the mismatched formation of disulfide bonds. The mutant enzyme in which all of the C-terminal four amino acid residues were replaced by alanine residues showed lower hydrolytic activity and an indistinguishable CD spectrum compared with the wild-type enzyme, and also recovered its activity from the denatured and reduced state by air oxidation. The D121 mutant enzymes showed decreased hydrolytic activity and identical CD spectra compared with the wild type. The recovery rates of activity of D121A and D121K were determined to be lower than that of the wild-type enzyme, while the rate of recovery of D121E was comparable to that of the wild type. The C-terminal amino acids play a significant role in the formation of the correct disulfide bonds during the refolding process, and the interaction of amino acid residues and the existence of the main chain around the C-terminal region are both important for achieving the efficient packing of the RNase A molecule.
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PMID:Significance of the four carboxyl terminal amino acid residues of bovine pancreatic ribonuclease A for structural folding. 1182 Sep 31

We have found that tissue-type transglutaminase (tTG), also called TGc, TGase2 and Galpha(h), has the activity of protein disulphide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. The PDI activity of tTG was strongly inhibited by bacitracin, which is a frequently used inhibitor of conventional PDI activity. It was substantially inhibited by the simultaneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least 200-fold excess did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in a fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalysed by a domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG completely abolished the transglutaminase activity, as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca(2+). It was not inhibited by nucleotides including GTP at all, unlike the other activity of tTG.
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PMID:A novel function of tissue-type transglutaminase: protein disulphide isomerase. 1273 32

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitro by changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as beta-ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivo in wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.
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PMID:RNase activity requires formation of disulfide bonds and is regulated by the redox state. 1560 66

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.
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PMID:Nonaggregating refolding of ribonuclease A using reverse micellar dialysis. 1562 75

PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV-visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent K(m) of PDI for diabz-GSSG was estimated to be approx. 15 muM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.
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PMID:A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching. 1596 Jun 11


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