Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.4 (SOR)
 720 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.
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PMID:Purification and characterization of monodehydroascorbate reductase from soybean root nodules. 172 43

Chlorella vulgaris contained only one isoform of ascorbate peroxidase (AsAP) as the hydrogen peroxide (H2O2)-scavenging system except for catalase at a specific activity of 3.3 +/- 0.2 units/mg protein. The activity of glutathione peroxidase was not detected in the extracts from cells grown in the absence and presence of sodium selenite. We detected the activity of monodehydroascorbate reductase involved in the regeneration of ascorbate, but we failed to detect the dehydroascorbate reductase activity. AsAP has been purified to electrophoretic homogeneity from Chlorella cells. The enzyme was a monomer with a molecular mass of 32 kDa using gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme showed higher specificity with ascorbate than with pyrogallol. The K(m) values of the enzyme for ascorbate and H2O2 were 111 +/- 8.9 and 20 +/- 2.5 microM, respectively. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. The enzyme was markedly inhibited by 0.2 mM p-chloromercuribenzoate. The enzyme cross-reacted by immunoblotting with the monoclonal antibody raised against Euglena cytosolic AsAP. The amino acid sequences in the N-terminal region of Chlorella AsAP showed no significant similarity to any other AsAPs from higher plants and algae.
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PMID:Purification and characterization of ascorbate peroxidase in Chlorella vulgaris. 967 48

The production of superoxide radicals (O2(-).) and the activities of ferricyanide reductase and cytochrome c reductase were investigated in peroxisomal membranes from pea (Pisum sativum L.) leaves using NADH and NADPH as electron donors. The generation of O2(-). by peroxisomal membranes was also assayed in native polyacrylamide gels using an in situ staining method with NitroBlue Tetrazolium (NBT). When peroxisomal membranes were assayed under native conditions using NADH or NADPH as inducer, two different O2(-).-dependent Formazan Blue bands were detected. Analysis by SDS/PAGE of these bands demonstrated that the NADH-induced NBT reduction band contained several polypeptides (PMP32, PMP61, PMP56 and PMP18, where PMP is peroxisomal membrane polypeptide and the number indicates molecular mass in kDa), while the NADPH-induced band was due exclusively to PMP29. PMP32 and PMP29 were purified by preparative SDS/PAGE and electroelution. Reconstituted PMP29 showed cytochrome c reductase activity and O2(-). production, and used NADPH specifically as electron donor. PMP32, however, had ferricyanide reductase and cytochrome c reductase activities, and was also able to generate O2(-). with NADH as electron donor, whereas NADPH was not effective as an inducer. The reductase activities of, and O2(-). production by, PMP32 were inhibited by quinacrine. Polyclonal antibodies against cucumber monodehydroascorbate reductase (MDHAR) recognized PMP32, and this polypeptide is likely to correspond to the MDHAR reported previously in pea leaf peroxisomal membranes.
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PMID:Characterization of membrane polypeptides from pea leaf peroxisomes involved in superoxide radical generation. 989 98

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.
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PMID:Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes. 1212 48

Efficient destruction of hydrogen peroxide (H(2)O(2)) in peroxisomes requires the action of an anti-oxidant defense system, which consists of low molecular weight anti-oxidant compounds, such as ascorbic acid, along with protective enzymes, such as catalase and ascorbate peroxidase (APX). We investigated the contribution of the ascorbate enzyme system to the consumptions of H(2)O(2) and NADH within glyoxysomes of germinating castor beans (Ricinus communis). We solubilized the glyoxysomal membrane APX (gmAPX) using octyl-glucoside and purified its activity by gel filtration. The activity was associated with a 34kDa protein, as determined by SDS-gel electrophoresis and Western blotting. The enzymatic properties of gmAPX were studied and this enzyme was found to utilize ascorbic acid as its most effective natural electron donor but it would also use pyrogallol and guaiacol at a smaller extent. Cyanide and azide drastically inhibited gmAPX, as well as certain thiol-modifying reagents and some metal chelators. The inhibition by cyanide and azide of the enzyme combined with its absorption spectra confirmed that it is a hemoprotein. The apparent K(m) value of the enzyme for ascorbic acid was 300 microM while the K(m) for H(2)O(2) was 60 microM. APX in the glyoxysomal membrane can work in cooperation with monodehydroascorbate reductase to oxidize NADH, regenerate ascorbate, detoxify H(2)O(2), and protect the integrity of glyoxysomal proteins and membranes.
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PMID:Ascorbate peroxidase, a scavenger of hydrogen peroxide in glyoxysomal membranes. 1563 24

Ascorbate peroxidase (APX) of the liverwort Pallavicinia lyelli was extracted and purified through ammonium sulfate precipitation, Butyl-Toyopearl, DEAE-Cellulofine and Sephadex G-75 chromatography. The purification factor for APX was 285 with 7.9% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of APX was approximately 28 kDa estimated by SDS-PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 6.0. The enzyme had a temperature optimum at 40 degrees C and was relatively stable at 60 degrees C, with 54% loss of activity. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity and the capacity to utilize alternate electron donors. p-Chloromercuribenzoate (pCMB), hydroxyurea and salicylic acid (SA) significantly inhibited APX activity. Ascorbate (AsA) and pyrogallol were found to be efficient substrates for Pallavicinia APX, considering the Vmax/Km ratio. We detected the activity of monodehydroascorbate reductase (MDHAR) involved in the regeneration of ascorbate, but failed to detect the dehydroascorbate reductase (DHAR) activity. The data obtained in this study may help to understand desiccation tolerance mechanism in the liverwort.
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PMID:Purification and kinetic characterization of the liverwort Pallavicinia lyelli (Hook.) S. Gray. cytosolic ascorbate peroxidase. 2061 66

Plants reconfigure their metabolic network under stress conditions. Changes of mitochondrial metabolism such as tricarboxylic acid (TCA) cycle and amino acid metabolism are reported in Arabidopsis roots but the exact molecular basis underlying this remains unknown. We here hypothesise the reassembly of enzyme protein complexes to be a molecular mechanism for metabolic regulation and tried in the present study to find out mitochondrial protein complexes which change their composition under oxidative stress by the combinatorial approach of proteomics and metabolomics. Arabidopsis seedlings were treated with menadione to induce oxidative stress. The inhibition of several TCA cycle enzymes and the oxidised NADPH pool indicated the onset of oxidative stress. In blue native/SDS-PAGE analysis of mitochondrial protein complexes the intensities of 18 spots increased and those of 13 spots decreased in menadione treated samples suggesting these proteins associate with, or dissociate from, protein complexes. Some spots were identified as metabolic enzymes related to central carbon metabolism such as malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, monodehydroascorbate reductase and alanine aminotransferase. The change in spot intensity was not directly correlated to the total enzyme activity and mRNA level of the corresponding enzyme but closely related to the metabolite profile, suggesting the metabolism is regulated under oxidative stress at a higher level than translation. These results are somewhat preliminary but suggest the regulation of the TCA cycle, glycolysis, ascorbate and amino acid metabolism by reassembly of plant enzyme complexes.
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PMID:Alteration of mitochondrial protein complexes in relation to metabolic regulation under short-term oxidative stress in Arabidopsis seedlings. 2114 42

Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.
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PMID:Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY. 2372 93