Partial Purification and Properties of Soluble Ascorbate Peroxidases From Pea Leaves

Gerbling, Klaus-Peter; Kelly, Grahame J.; Fischer, Kurt-H.; Latzko, Erwin

doi:10.1016/s0176-1617(84)80051-6

Cited by 87 publications

(39 citation statements)

References 14 publications

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“…Ascorbate peroxidase activity was measured spectrophotometrically following the oxidation of ascorbate in the presence of H202 (Gerbling et al, 1984). Glutathio7e reductase activity was determined spectrophotometrically following oxidation of NADPH in the presence of oxidized glutathione (Foyer et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves

et al. 1994

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Protoplasts, vacuoles, and chloroplasts were isolated from leaves of 8-d-old barley (Hordeum vulgare 1. CY Cerbel) seedlings. Transport of ascorbate and dehydroascorbate into protoplasts and vacuoles was investigated. Contents of ascorbic acid, glutathione, and a-tocopherol and ascorbate peroxidase activity and glutathione reductase activity were analyzed in protoplasts, vacuoles, and chloroplasts. Uptake of ascorbate and dehydroascorbate by protoplasts showed saturation kinetics (K, = 90 WM reduced ascorbic acid, 20 p~ dyhydroascorbic acid). Effects of various membrane transport inhibitors suggested that transport was carrier mediated and driven by a proton electrochemical gradient. Translocation of ascorbate and dehydroascorbate into vacuoles did not show saturation kinetics. Neither was it influenced by effectors or by ATP but only by Mg2+, suggesting that translocation did not occur by carrier. Ascorbic acid was predominantly localized in the cytosol. Contents in the chloroplasts and vacuoles were low. The results are consistent with the view that ascorbate is synthesized in the cytosol and released to chloroplasts, apoplast, and vacuole following a concentration gradient. Translocation from the apoplast into the cytosol is against a steep gradient and appears to control the concentration of ascorbic acid in the apoplast. In its function as an antioxidant, aicorbate in the apoplast may be oxidized to dehydroascorbate, which can be efficiently transported back into the cytosol for regeneration to ascorbate.

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Section: Methodsmentioning

confidence: 99%

Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves

et al. 1994

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“…Crude extracts contained approximately 30% higher ascorbate peroxidase activities than extracts after gel fitration in the presence of ascorbate. This might indicate either an inactivation despite the presence of ascorbate or the loss of a low mol wt component with ascorbate peroxidase activity similar to that detected in pea extracts (12). The interference of catalase with the ascorbate peroxidase assay was in the range of 10% and was avoided by addition of aminotriazole (500 gM) to the assay mixture.…”

Section: Properties Of H202-scavening Enzymes In Spruce Needlesmentioning

confidence: 96%

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Polle¹,

Chakrabarti²,

Schürmann³

et al. 1990

Plant Physiol.

242

167

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Hydrogen peroxide (H202) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 500C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent Km = 40 millimolar) and H202 (apparent Km = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed. Plants are exposed to hydrogen peroxide (H202) from both external and internal sources. In the environment, H202 is photochemically produced in the atmosphere and is found in concentrations up to 100 AM in rain, clouds, and fog (15). On the cellular level, H202 is generated as a metabolite ofenzymic reactions and during photosynthesis (24

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“…AP has been found in higher plants [ 16], Chlamydomonas [23], Euglena [33], Trypanosoma [3] and some cyanobacteria [23,39]. The presence of its isozymes has been demonstrated in pea [8], spinach [25,38] and tea [4]. Chloroplastic AP has been well characterized, and proved to be a component of the scavenging system of active oxygens inevitably photoproduced in chloroplasts [9, The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X59600.…”

Section: Introductionmentioning

confidence: 99%

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

et al. 1992

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A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage lambda gt11 library of cDNA from Arabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)+ RNA from leaves of A. thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence of Arabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP of A. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochrome c peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochrome c peroxidase are conserved in the amino acid sequence of Arabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3' untranslated region of the cDNA.

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Partial Purification and Properties of Soluble Ascorbate Peroxidases From Pea Leaves

Cited by 87 publications

References 14 publications

Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves

Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

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