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)

A method is described for purification of sulfhydryl oxidase from bovine milk which consistently yields preparations with greater than 3000-fold purification over skim milk. A concentration-dependent association-dissociation of the enzyme was adapted to the development of an isolation procedure. Purified preparations exhibited two zones, both of which displayed activity, upon polyacrylamide disc gel electrophoresis, but only one zone following disc gel electrophoresis in sodium dodecyl sulfate. Its mobility indicated a subunit weight of 89,000. Several lines of evidence suggest that iron is an integral part of the enzyme. Treatment of the enzyme with EDTA resulted in complete loss of activity which could be subsequently restored by dialysis against 1 muM ferrous sulfate. Furthermore, atomic absorption analysis and neutron activation analysis of separate enzyme preparations each indicated 0.5 atom of iron per subunit. Chemical analyses of sulfhydryl oxidase accounted for 97% of the sample weight, of which 89% could be attributed to amino acid residues and 11% to carbohydrate residues. Five half-cystine residues per subunit were indicated by cysteic acid analysis and by sulfhydryl group determination following reaction with sodium borohydride. Comparison of this value to the total sulfhydryl groups without reduction tentatively suggests the presence of one disulfide bond. Sulfhydryl oxidase was found to catalyze the oxidation of sulfhydryl groups in both small compounds and proteins, using O2 as oxidant and producing, in equimolar quantities, H2O2 and the corresponding disulfide. A Michaelis constant of 90 muM was obtained using reduced glutathione as substrate, under conditions of optimal pH and temperature, viz., pH 7.0 and 35 degrees. Substrate inhibition was apparent at GSH concentrations above 0.8 mM. In the presence of sulfhydryl oxidase, reductively denatured RNase was reoxidized and fully reactivated within 1 hour, whereas in the absence of the oxidase under otherwise identical conditions, full recovery of RNase activity required 24 hours. The presence of reducing agent was not required for this activity, nor was prior reduction of the sulfhydryl oxidase. Based on the observed activity, it appears that the enzyme could be involved in the biosynthesis of disulfide bonds in certain proteins.
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PMID:Isolation and characterization of sulfhydryl oxidase from bovine milk. 112 23

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.
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PMID:Purification and properties of sulfhydryl oxidase from bovine pancreas. 304 13

Sulfhydryl oxidase isolated from bovine skim milk membrane vesicles catalyzes de novo formation of disulfide bonds with the substrates cysteine, cysteine-containing peptides, and reduced proteins using molecular oxygen as the electron acceptor. Initial rates for sulfhydryl oxidase-catalyzed oxidation of reduced ribonuclease exhibited typical Michaelis-Menten kinetics at low substrate concentrations. Substrate inhibition of the oxidative activity was observed at ribonuclease concentrations greater than 40 microM, similar to that observed with reduced glutathione or other small thiol substrates. The inhibition was more pronounced when ribonuclease activity was used to monitor the rates, presumably due to concentration-dependent formation of nonnative disulfide bonds. Thus, a maximum in the rate of regain of ribonuclease activity was observed at a 40 microM concentration, while optimum recovery was observed at 30 microM. The Michaelis constant obtained with reduced ribonuclease is 17.4 microM which corresponds to a sulfhydryl concentration of 0.14 mM, a value that compares favorably with the best small thiol substrate, reduced glutathione. Disulfide-containing intermediates in the oxidation pathway, as determined by ion-exchange chromatography of alkylated reaction mixtures, appeared to be similar for air oxidation and enzyme-catalyzed oxidation of the protein. The pH optimum, tissue location, and kinetic characteristics of sulfhydryl oxidase are compatible with a suggested physiological function of direct catalysis of disulfide bond formation in secretory proteins or indirect participation through provision of oxidized glutathione for protein disulfide-isomerase-catalyzed thiol/disulfide interchange.
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PMID:Sulfhydryl oxidase-catalyzed formation of disulfide bonds in reduced ribonuclease. 366 39

During the refolding and oxidation of reductively denatured ribonuclease A in solution, there is a marked lag in appearance of enzymatic activity as compared to the oxidation of sulfhydryl groups, whether such oxidation is spontaneous or is catalyzed by sulfhydryl oxidase. However, if ribonuclease is covalently attached to a derivatized glass surface, a lag period is not observed during the reformation of native structure from the completely reduced, denatured state. These results suggest that, in solution, intermolecular interactions alter the pathway of polypeptide chain folding and disulfide bond formation, leading to nonnative disulfides which do not rapidly interchange to form native pairings. The isolation of refolding polypeptide chains by covalent immobilization prevents such interactions. Presumably, such intermolecular interactions would be similarly prevented by "isolation" of nascent polypeptide chains during protein synthesis on ribosomes.
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PMID:Renaturation of soluble and immobilized ribonuclease: are the polypeptide folding pathways for structure formation the same for soluble proteins and for proteins associated with a surface? 400 93

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

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.
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PMID:Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides. 1042 77

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.
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PMID:Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes. 1217 51

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX family members contain several domains, including an N-terminal thioredoxin domain (Trx) and an FAD-binding-domain (ERV) toward the C-terminus. Partial proteolysis of avian QSOX leads to two fragments, designated 30 and 60 kDa from their apparent mobilities on SDS-PAGE. The 30 kDa fragment is a monomer under nondenaturing conditions and contains a Trx domain with a CxxC sequence typical of protein disulfide isomerase (WCGHC). This QSOX fragment is not detectably glycosylated, contains no detectable FAD, and shows undetectable sulfhydryl oxidase activity. In contrast, the 60 kDa fragment is a dimeric glycoprotein that binds FAD tightly and oxidizes dithiothreitol about 1000-fold slower than intact QSOX. Reduced RNase is not a significant substrate of the 60 kDa fragment. The redox behavior of the 60 kDa flavoprotein fragment is profoundly different from that of intact QSOX. Thus, dithionite or photochemical reduction of the 60 kDa fragment leads to two-electron reduction of the FAD without subsequent reduction of the other two CxxC motifs or the appearance of a thiolate to flavin charge-transfer complex. Further characterization of the fragments and insights gained from the crystal structure of yeast ERV2p (Gross, E., Sevier, C. S., Vala, A., Kaiser, C. A., and Fass, D. (2002) Nat. Struct. Biol. 9, 61-67) suggest that the flow of reducing equivalents in intact avian QSOX is dithiol substrate --> C80/83 --> C519/522 --> C459/462 --> FAD --> oxygen. The ancient fusion of thioredoxin domains to a catalytically more limited ERV domain has produced an efficient catalyst for the direct introduction of disulfide bonds into a wide range of proteins and peptides in multicellular organisms.
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PMID:Inter-domain redox communication in flavoenzymes of the quiescin/sulfhydryl oxidase family: role of a thioredoxin domain in disulfide bond formation. 1269 53

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.
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PMID:Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme. 1297 44

Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases.
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PMID:A flavin-dependent sulfhydryl oxidase in bovine milk. 1794 90


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