Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
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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

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

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