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1.
The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. 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. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves. 相似文献
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To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrane proteins of glyoxysomes. One of them was identified as an ascorbate peroxidase (pAPX) that is localized on glyoxysomal membranes. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg(2+), indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure. 相似文献
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The sorting signals for peroxisomal membrane-bound ascorbate peroxidase are within its C-terminal tail 总被引:1,自引:0,他引:1
Peroxisomal ascorbate peroxidase (APX) is a carboxyl tail-anchored, type II (N(cytosol)-C(matrix)) integral membrane protein that functions in the regeneration of NAD(+) in glyoxysomes of germinated oilseeds and protection of peroxisomes in other organisms from toxic H(2)O(2). Recently we showed that cottonseed peroxisomal APX was sorted post-translationally from the cytosol to peroxisomes via a novel reticular/circular membranous network that was interpreted to be a subdomain of the endoplasmic reticulum (ER), named peroxisomal ER (pER). Here we report on the molecular signals responsible for sorting peroxisomal APX. Deletions or site-specific substitutions of certain amino acid residues within the hydrophilic C-terminal-most eight-amino acid residues (includes a positively charged domain found in most peroxisomal integral membrane-destined proteins) abolished sorting of peroxisomal APX to peroxisomes via pER. However, the C-terminal tail was not sufficient for sorting chloramphenicol acetyltransferase to peroxisomes via pER, whereas the peptide plus most of the immediately adjacent 21-amino acid transmembrane domain (TMD) of peroxisomal APX was sufficient for sorting. Replacement of the peroxisomal APX TMD with an artificial TMD (devoid of putative sorting sequences) plus the peroxisomal APX C-terminal tail also sorted chloramphenicol acetyltransferase to peroxisomes via pER, indicating that the peroxisomal APX TMD does not possess essential sorting information. Instead, the TMD appears to confer the proper context required for the conserved positively charged domain to function within peroxisomal APX as an overlapping pER sorting signal and a membrane peroxisome targeting signal type 2. 相似文献
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Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase 总被引:22,自引:0,他引:22
A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site. 相似文献
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Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves 总被引:13,自引:0,他引:13
The degradation of peroxisomal and nonperoxisomal proteins by endoproteases of purified peroxisomes from senescent pea (Pisum sativum L.) leaves has been investigated. In our experimental conditions, most peroxisomal proteins were endoproteolytically degraded.
This cleavage was prevented, to some extent, by incubation with 2 mM phenylmethylsulfonylfluoride, an inhibitor of serine
proteinases. The peroxisomal enzymes glycolate oxidase (EC 1.1.3.1), catalase (EC 1.11.1.6) and glucose-6-phosphate dehydrogenase
(EC 1.1.1.49) were susceptible to proteolytic degradation by peroxisomal endoproteases, whereas peroxisomal manganese superoxide
dismutase (EC 1.15.1.1) was not. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from spinach and urease (EC
3.5.1.5) from jack bean were strongly degraded in the presence of peroxisomal matrices. These results indicate that proteases
from plant peroxisomes might play an important role in the turnover of peroxisomal proteins during senescence, as well as
in the turnover of proteins located in other cell compartments during advanced stages of senescence. On the other hand, our
data show that peroxisomal endoproteases could potentially carry out the partial proteolysis which results in the irreversible
conversion of xanthine dehydrogenase into the superoxide-generating xanthine oxidase (EC 1.1.3.22). This suggests a possible
involvement of the peroxisomal endoproteases in a regulated modification of proteins.
Received: 25 January 1999 / Accepted: 3 June 1999 相似文献
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Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress. 总被引:15,自引:0,他引:15
The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme. 相似文献
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Romero-Puertas MC Corpas FJ Sandalio LM Leterrier M Rodríguez-Serrano M Del Río LA Palma JM 《The New phytologist》2006,170(1):43-52
The glutathione reductase (GR; EC 1.6.4.2) isozyme present in peroxisomes has been purified for the first time, and its unequivocal localization in these organelles, by immunogold electron microscopy, is reported. The enzyme was purified c. 21-fold with a specific activity of 9523 units mg(-1) protein, and a yield of 44 microg protein kg(-1) leaves was obtained. The subunit size of the peroxisomal GR was 56 kDa and the isoelectric point was 5.4. The enzyme was recognized by a polyclonal antibody raised against total GR from pea (Pisum sativum) leaves. The localization of GR in peroxisomes adds to chloroplasts and mitochondria where GR isozymes are also present, and suggests a multiple targeting of this enzyme to distinct cell compartments depending on the metabolism of each organelle under the plant growth conditions. The expression level of GR in several organs of pea plants and under different stress conditions was investigated. The possible role of peroxisomal GR under abiotic stress conditions, such as cadmium toxicity, high light, darkness, high temperature, wounding and low temperature, is discussed. 相似文献
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Narendra S Venkataramani S Shen G Wang J Pasapula V Lin Y Kornyeyev D Holaday AS Zhang H 《Journal of experimental botany》2006,57(12):3033-3042
Ascorbate peroxidase (APX) exists as several isoforms that are found in various compartments in plant cells. The cytosolic and chloroplast APXs appear to play important roles in antioxidation metabolism in plant cells, yet the function of peroxisomal APX is not well studied. In this study, the localization of a putative peroxisomal membrane-bound ascorbate peroxidase, APX3 from Arabidopsis, was confirmed by studying the green fluorescent protein (GFP)-APX3 fusion protein in transgenic plants. GFP-APX3 was found to co-localize with a reporter protein that was targeted to peroxisomes by the peroxisomal targeting signal 1. The function of APX3 in Arabidopsis was investigated by analysing an APX3 knockout mutant under normal and several stress conditions. It was found that loss of function in APX3 does not affect Arabidopsis growth and development, suggesting that APX3 may not be an important antioxidant enzyme in Arabidopsis, at least under the conditions that were tested, or the function of APX3 could be compensated by other antioxidant enzymes in plant cells. 相似文献
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An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization 总被引:1,自引:0,他引:1
Kumaresan Kavitha Gayatri Venkataraman Ajay Parida 《Plant Physiology and Biochemistry》2008,46(8-9):794-804
APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements. 相似文献
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Mitochondrial and peroxisomal metabolism of glutaryl-CoA 总被引:1,自引:0,他引:1
Using a fraction purified from liver peroxisomes, we demonstrate that products of the glutaryl-CoA oxidase reaction are glutaconyl-CoA and H2O2. No glutaconyl-CoA decarboxylation occurs with this fraction. In whole tissue homogenates, the handling of glutaryl-CoA by glutaryl-CoA dehydrogenase is inhibited when reoxidation of FADH2 is blocked. Under these conditions, glutaconyl-CoA decarboxylation, however, can still occur and 14CO2 is produced from labelled glutaryl-CoA in mole/mole ratio with H2O2. These data indicate that in the absence of its mitochondrial dehydrogenation, glutaryl-CoA is oxidized in peroxisomes to glutaconyl-CoA which is probably transferred to mitochondria where it is decarboxylated and further processed. This hypothesis allows coherent explanation for the observed organic aciduria in both glutaricaciduria types I and II. 相似文献
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Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each other's activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate. 相似文献
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ABSTRACT Sixteen species of Gymnosperms have been screened for cytosolic ascorbate peroxidase by means of native polyacrylamide gel electrophoresis. This analysis shows that a single form of the enzyme is the most common situation. The enzyme reveals a similar electrophoretic mobility in species belonging to the same genus and sometimes to different genera. In some Pinaceae, two bands of activity were observed. The presence in the archaic spermatophyte Ginkgo biloba, as well as in the more advanced monocotyledons, of three isoforms of ascorbate peroxidase, might suggest that three different cytosolic ascorbate peroxidase genes were already present in this archaic species. 相似文献
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Regulation and function of ascorbate peroxidase isoenzymes 总被引:59,自引:0,他引:59
Shigeoka S Ishikawa T Tamoi M Miyagawa Y Takeda T Yabuta Y Yoshimura K 《Journal of experimental botany》2002,53(372):1305-1319
Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses. 相似文献
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Effects of plant o-dihydroxyphenols on ascorbate (ASA) content and ascorbate peroxidase (APOX) activity in the tissues of the grain aphid Sitobion avenae and the bird cherry-oat aphid Rhopalosiphum padi were studied. Among the aphid morphs, the highest ASA content and APOX activity were noted for larvae and the lowest for
wingless apterae. When exposed to o-dihydroxyphenols, aphids of both species contained significantly lower concentrations of ASA and higher APOX activity than
the controls. Among the studied compounds, caffeic acid had the strongest effect on ASA-based antioxidant responses in that
caffeic acid caused a 5-fold decrease of ASA in aphid tissues. The influences of the plant o-dihydroxyphenols on antioxidant defense mechanisms within the cereal aphid species are discussed. 相似文献
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Efimov I Papadopoulou ND McLean KJ Badyal SK Macdonald IK Munro AW Moody PC Raven EL 《Biochemistry》2007,46(27):8017-8023
Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees '(compound I/compound II) and E degrees '(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes. 相似文献
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Light-determined activation of ferments of ascorbate-glutation cycle, ascorbate-oxidase in chloroplasts and cytosol is demonstrated as well as ascorbate-peroxidase, monodehydroascorbate-reductase, glutation-reductase and ascorbate-oxydase in mitochondria. On the other hands activity of mitochondrial dehydroascorbate-reductase increased on reduction of light most likely due to function of electron transport from glutation to dehydroascorbate in mitochondria. Glutation metabolism is proved to be endogenic catalytic process where the amount reconstructed glutation changes slowly with a delay and gradually follow light changes. Light dependable changes of glutation content in chloroplasts ensure resistance of ferment system again hydrogen peroxide and superoxide radicals that generate intensively at light. 相似文献