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PubMed2000

Antisense suppression of 2-cysteine peroxiredoxin in Arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism.

The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool.

Antioxidants↗
PubMed1998

Purification and characterization of ascorbate peroxidase in Chlorella vulgaris.

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.

Amino Acid Sequence↗
PubMed2003

Antioxidant systems and their relationship with the response of pepper fruits to storage at 20 degrees C.

Fresh peppers (Capsicum annuum L., variety California) in their green and red ripe stages were stored at 20 degrees C for 7 and 19 days to determine the effects of storage on whole fruit antioxidant capacity (TAA) and ascorbate (ASC) content, as well as on some antioxidant enzyme activities, such as catalase (CAT), superoxide dismutase (SOD), and those of the ASC-glutathione cycle. At least one Mn-SOD, two Fe-SODs, and three CuZn-SODs were detected in the fruit extract after native polyacrylamide gel electrophoresis. All of the SOD isozymes and glutathione reductase had higher activity levels in the red control fruits than in the green fruits, whereas the activities of monodehydroascorbate and dehydroascorbate reductase were higher in green fruits. Ascorbate peroxidase (APX) was found to be similar in both fruits. SODs, CAT, and APX seem to be involved in pepper fruit ripening and senescence during storage at 20 degrees C, perhaps influencing the active oxygen species levels in the fruit. TAA, as well as the ASC content, was higher in red peppers than in green, and storage increased the ASC in both green and red fruits.

Antioxidants↗
PubMed2005

Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress.

Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance.

Arabidopsis↗
PubMed2005

Duodenal ascorbate and ferric reductase in human iron deficiency.

BACKGROUND: The first step in iron absorption requires the reduction of ferric iron to ferrous iron, a change that is catalyzed by duodenal ferric reductase. Iron deficiency is associated with high iron absorption, high ferric reductase activity, and high duodenal ascorbate concentrations in experimental animals, but it is not known whether a relation between reductase and ascorbate is evident in humans. OBJECTIVE: The objective of the study was to assess the relation between ferric reductase activity in human duodenal biopsy specimens and ascorbate concentrations in iron-replete and iron-deficient subjects. DESIGN: Patients and control subjects were overnight-fasted adults presenting sequentially for upper gastrointestinal endoscopic investigation. Ferric reductase activity in duodenal biopsy specimens was assayed by using nitroblue tetrazolium. Ascorbate was assayed in duodenal biopsy specimens and plasma. RESULTS: Iron-deficient patients had significantly higher reductase activity (n = 6-9; P < 0.05) and duodenal (n = 20; P < 0.001) and plasma (n = 6; P < 0.001) ascorbate concentrations than did control subjects. Incubation of biopsy specimens with dehydroascorbate (to boost cellular ascorbate) increased reductase activity in the tissues that initially had normal activity (n = 9; P < 0.01) but inhibited reductase activity in the tissues that already had high reductase activity (n = 13; P < 0.001). CONCLUSIONS: Iron deficiency in humans is associated with increased duodenal ascorbate concentrations. This finding suggests that increased reductase activity is partly due to an increase in this substrate for duodenal cytochrome b reductase 1.

Adult↗
PubMed2003

Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants.

Key components of the ascorbate-glutathione cycle in Arabidopsis cell organelles are encoded by single organellar targeted isoforms that are dual localized in the chloroplast stroma and the mitochondrion. We demonstrate the presence of the ascorbate-glutathione cycle in purified Arabidopsis mitochondria using enzymatic activity, proteomic and in vitro and in vivo subcellular targeting data that identify the gene products responsible. In vitro experiments using a dual import assay assessing mitochondrial and chloroplast imports simultaneously show dual targeting of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase gene products to mitochondria and chloroplasts, while a putative dehydroascorbate reductase protein is only imported into mitochondria. In vivo subcellular localization using green fluorescent protein fusion proteins show clear targeting of all gene products to mitochondria. Transcript levels show these genes are induced by oxidative chemical stresses targeted to chloroplasts and/or mitochondria and are elevated during photosynthetic operation in the light. Together these data present a model of an integrated ascorbate-glutathione antioxidant defense common to plastids and mitochondria that is linked at the level of the genome in Arabidopsis.

Antioxidants↗
PubMed1998

Changes in salicylic acid and antioxidants during induced thermotolerance in mustard seedlings

Heat-acclimation or salicylic acid (SA) treatments were previously shown to induce thermotolerance in mustard (Sinapis alba L.) seedlings from 1.5 to 4 h after treatment. In the present study we investigated changes in endogenous SA and antioxidants in relation to induced thermotolerance. Thirty minutes into a 1-h heat-acclimation treatment glucosylated SA had increased 5.5-fold and then declined during the next 6 h. Increases in free SA were smaller (2-fold) but significant. Changes in antioxidants showed the following similarities after either heat-acclimation or SA treatment. The reduced-to-oxidized ascorbate ratio was 5-fold lower than the controls 1 h after treatment but recovered by 2 h. The glutathione pool became slightly more oxidized from 2 h after treatment. Glutathione reductase activity was more than 50% higher during the first 2 h. Activities of dehydroascorbate reductase and monodehydroascorbate reductase decreased by at least 25% during the first 2 h but were 20% to 60% higher than the control levels after 3 to 6 h. One hour after heat acclimation ascorbate peroxidase activity was increased by 30%. Young leaves appeared to be better protected by antioxidant enzymes following heat acclimation than the cotyledons or stem. Changes in endogenous SA and antioxidants may be involved in heat acclimation.

Journal Article↗
PubMed2001

Subcellular localization of a glutathione-dependent dehydroascorbate reductase within specific rat brain regions.

Recently, we described the occurrence of a dehydroascorbate reductase within the rat CNS. This enzyme regenerates ascorbate after it is oxidized during normal aerobic metabolism. In this work, we describe the neuronal compartmentalization of the enzyme, using transmission electron microscopy of those brain areas in which the enzyme was most densely present when observed under light microscopy. In parallel biochemical studies, we performed immunoblotting and measured the enzyme activity of the cytoplasm and different nuclear fractions. Given the abundance of ascorbate in the caudate-putamen, we focused mostly on the occurrence of dehydroascorbate reductase at the striatal subcellular level. We also studied cerebellar Purkinje cells, hippocampal CA3 pyramidal cells and giant neurons in the magnocellular part of the red nucleus. In addition to neurons, immunolabeling was found in striatal endothelial cells, in the basal membrane of blood vessels and in perivascular astrocytes. In neuronal cytosol, the enzyme was observed in a peri-nuclear position and on the nuclear membrane. In addition, in both the striatum and the cerebellum, we found the enzyme within myelin sheets. Dehydroascorbate reductase was also present in the nucleus of neurons, as further indicated by measuring enzyme activity and by immunoblotting selected nuclear fractions. Immunocytochemical labeling confirmed that the protein was present in isolated pure nuclear fractions. Given the great amount of free radicals which are constantly generated in the CNS, the discovery of a new enzyme with antioxidant properties which translocates into neuronal nuclei appears to be a potential starting point to develop alternative strategies in neuroprotection.

Animals↗
PubMed1994

Ascorbate and exercise in the Northern Ireland population.

The relationship between physical activity, physical fitness and serum ascorbate was examined in the Northern Ireland Health and Activity Survey. This was a cross sectional population study (n = 1600), using a two stage probability sample, of the population of N. Ireland. Physical activity profile was recorded by computer assisted interview and physical fitness was determined by estimation of VO2 max by extrapolation from submaximal oxygen uptake. Serum ascorbate was measured using a colorimetric reaction of 2,4 dinitrophenylhydrazine with dehydroascorbate. Mean serum ascorbate was greater in females than males (p < 0.001), and was lower in smokers than non-smokers in both males (p = 0.01) and females (p < 0.001). There was no statistically significant relationship between serum ascorbate and age, social class, body mass index, physical activity or physical fitness in males but there was a relationship with age (p < 0.01) and physical fitness (p < 0.05) in females.

Adolescent↗
PubMed1986

Hydroperoxide metabolism in cyanobacteria.

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.

Ascorbate Peroxidases↗
PubMed2004

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation.

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.

Ascorbate Oxidase↗
PubMedDate not supplied

[Parameters of oxidative stress in the lens, aqueous humor and blood in patients with diabetes and senile cataracts].

INTRODUCTION: Contemporary hypothesis considers the oxidative stress as a crucial event in age-related processes in the body, as well as in the age-related cataract formation. Disturbance of balance between oxidative processes and antioxidative defenses causes the oxidative stress that can damage proteins, lipids, polysaccharides and nucleic acids. The secondary aging factors accelerate ageing processes. One of them is diabetes. It's numerous consequences that appear in the eye are well known. PURPOSE: The purpose of this paper is to analyze influence of the non-insulin-dependent diabetes (Type II) on cataract genesis mainly through action of the oxidative stress and antioxidative defense. MATERIAL AND METHODS: Here were analyzed contents of the lipid oxidation products (lipid peroxides--LP) and total sulfhydryle groups (TSH) in the lens' corticonuclear blocks and antioxidative capacity in their humour aqueous expressed as percent of induced malondyaldehyde (% iMDA) in 14 samples obtained from patients with cataract and diabetes mellitus type II (without diabetic complications; duration of diabetes was 0-17 years) and compared to 66 samples of patients with cataract without diabetes, as well as some parameters of the oxidative stress in serums (content of vitamin C, acrobat--A, dehydroascorbate--DA and their relation, vitamin E, glutathione--GSH, peroxidase--P and catalase--Cat activity, content of malondyaldehyde--MDA and % iMDA) of 27 patients with age-related cataract and diabetes mellitus type II (without complications; same duration), and compared to the other 135 age-related cataract patients. Data were analyzed by multivariant and univariant analysis of variance (multivariant t-test, MANOVA and ANOVA). Also were analyzed frequencies of the secondary serium diseases in a clinical group of 162 patients with cataract and sex and age matched 55 examined people without cataract, as a control group, by a logistic regression. RESULTS: Patients with diabetes and cataract have lower values of almost all investigated parameters of antioxidative defense in their serum and higher level of the lipid peroxidation products (multivariant: p > 0.05). Level of glutathione in their serums is significantly lower (t = 2.27; p = 0.028). Intensity of lipid peroxidation in corticonuclear lens blocks is higher in patients with diabetes, whereas their total sulfhydryle groups and % iMDA in humour aqueous shows lower antioxidant capacity in the same group, probably because of higher intensity of oxidative stress, although not significantly (T = 2.23; F = 0.95; p > 0.05; univariant: p > 0.05). Also, by investigation of frequencies of the secondary ageing diseases in patients with age-related cataract and age and sex matched control subjects, by a logistic regression was found high odds ratio for diabetes (?2 = 18.87; p = 0.0044; const = 0.379; OR = 2.506). DISCUSSION: Statistically lower level of the serum glutathione in diabetic patients is important index of higher level of oxidative stress in their organism. Values of other investigated parameters of oxidative stress and antioxidative defense point to the some process. However, serum's parameters are not quite specific to the cataract, because active homeostatic mechanisms at the level of hematocamerular barrier and lens' metabolism could maintain local environment to the some extent. Yet, importance of glutathione level and metabolism of the eye's lens is great. Similar meaning bear the other sulfhydrile groups, i.e. of the lens' proteins-crystallines. So, total sulfhydrile groups level is good mark for the lens antioxidative defense, as well as the lipid peroxides are good mark for the intensity of oxidative processes. Percent of induced MDA in humour aqueous may be considered as the rest of antioxidative defense, similar to it's meaning in serum. Intensity of the oxidative stress in local compartment in diabetic patients in this paper is higher, compared to other age-related cataract patients without diabetes. Lack of significance could be explained by mentioned characteristics of the group (i.e. diabetics without complications), compared with other authors results, whose examined patients have had proliferative diabetic retinopathy. The odds ratio value of diabetes to the age-related cataract also confirms that diabetes is a risk factor for the age-related cataract genesis. CONCLUSION: Results confirm hypothesis of the oxidative stress role in the age-related cataract genesis, especially in patients with diabetes mellitus. Parameters of their antioxidative defense are lower and those of oxidative stress are higher in serums and in the lenses and humour aqueous. They also point to the dynamics and diversity of oxidative stress and antioxidative defense in different compartments of the body, as well as their different meaning in distinct physiological and pathological conditions.

Aged↗
PubMed1987

Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts.

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstrate a 2-3-fold increase in the turnover rate for ghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine beta-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.

Adenosine Triphosphate↗
PubMed1974

Uptake and reduction of oxidized and reduced ascorbate by human leukocytes.

Incubation of human leukocytes with dehydroascorbate (DHA) results in an increase in their reduced ascorbate (AA) content and hexose monophosphate shunt (HMS) activity, independent of oxygen supply. Incubation with AA induces these changes only in the presence of oxygen. The increase in HMS activity observed as cell AA increases by 1 micromol is the same during incubation with either DHA or AA. We propose that human leukocytes take up ascorbate as DHA (AA after oxidation to DHA) and reduce it promptly to AA, and that HMS stimulation upon incubation with either AA or DHA is a result of DHA reduction.

Aerobiosis↗
PubMed2005

Regulatory and functional interactions of plant growth regulators and plant glutathione S-transferases (GSTs).

Plant glutathioneS-transferases (GSTs) are a heterogeneous superfamily of multifunctional proteins, grouped into six classes. The tau (GSTU) and phi (GSTF) class GSTs are the most represented ones and are plant-specific, whereas the smaller theta (GSTT) and zeta (GSTZ) classes are also found in animals. The lambda GSTs (GSTL) and the dehydroascorbate reductases (DHARs) are more distantly related. Plant GSTs perform a variety of pivotal catalytic and non-enzymatic functions in normal plant development and plant stress responses, roles that are only emerging. Catalytic functions include glutathione (GSH)-conjugation in the metabolic detoxification of herbicides and natural products. GSTs can also catalyze GSH-dependent peroxidase reactions that scavenge toxic organic hydroperoxides and protect from oxidative damage. GSTs can furthermore catalyze GSH-dependent isomerizations in endogenous metabolism, exhibit GSH-dependent thioltransferase safeguarding protein function from oxidative damage and DHAR activity functioning in redox homeostasis. Plant GSTs can also function as ligandins or binding proteins for phytohormones (i.e., auxins and cytokinins) or anthocyanins, thereby facilitating their distribution and transport. Finally, GSTs are also indirectly involved in the regulation of apoptosis and possibly also in stress signaling. Plant GST genes exhibit a diversity of expression patterns during biotic and abiotic stresses. Stress-induced plant growth regulators (i.e., jasmonic acid [JA], salicylic acid [SA], ethylene [ETH], and nitric oxide [NO] differentially activate GST gene expression. It is becoming increasingly evident that unique combinations of multiple, often interactive signaling pathways from various phytohormones and reactive oxygen species or antioxidants render the distinct transcriptional activation patterns of individual GSTs during stress. Underestimated post-transcriptional regulations of individual GSTs are becoming increasingly evident and roles for phytohormones (i.e., ABA and JA) in these processes are being anticipated as well. Finally, indications are emerging that NO may regulate the activity of specific plant GSTs. In this review, the current knowledge on the regulatory and functional interactions of phytohormones and plant GSTs are covered. We refer to a previous extensive review on plant GSTs (Marrs, 1996) for most earlier work. An introduction on the classification and roles of plant GSTs is included here, but these topics are more extensively discussed in other reviews (Dixon et al., 2002a; Edwards et al., 2000; Frova, 2003).

Gene Expression↗
PubMed1998

Oxygen-accepting antioxidants which arise during ascorbate oxidation.

To identify potential antioxidant compounds derived from ascorbate, the hydrogen peroxide-induced oxidation of ascorbate and dehydroascorbate was studied by gas-chromatography electron impact mass spectrometry and liquid-chromatography electrospray mass spectrometry in real time. Significant differences in ascorbate and dehydroascorbate oxidation occurred at pH 3.3 compared to pH 7.4. Of note, the primary species present in dehydroascorbate (DHA)-containing solutions at pH 7.4 had a spectrum consistent with diketogulonate. Hydrogen peroxide exposure of DHA-containing solutions formed threonate more rapidly at pH 7.4 than at pH 3.3. In these solutions, a 5-carbon species with mass spectral characteristics of a 3,4,5-trihydroxy-2-ketopentanoate appeared to be an intermediate between diketogulonate and threonate, and was more labile than other species in the presence of hydrogen peroxide. These data suggest that a 3,4,5-trihydroxy-2-ketopentanoate is potentially a key antioxidant compound in the ascorbate degradation cascade and in ascorbate-containing solutions at physiologic pH.

Antioxidants↗
PubMedDate not supplied

lambda-crystallin related to dehydroascorbate reductase in the rabbit lens.

PURPOSE: To evaluate the relationship of lambda-crystallin to reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydroascorbate (DHA) reductase found specifically in the rabbit lens. METHODS: DHA reductase Fractions I-IV were separated from the lambda/betaL1-crystallin fraction of rabbit lens soluble protein by diethylaminoethyl (DEAE)-cellulose ion-exchange column chromatography, and then the enzyme was partially purified from Fraction II by rechromatography on the same ion-exchange column. The isolated DHA reductase fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, native isoelectric focusing and two-dimensional gel electrophoresis. RESULTS: Using Western blot and a probe of antiserum to recombinant lambda-crystallin, the main 33-kDa protein band was strongly stained in all the rabbit lens DHA reductase fractions, and most of the additional protein bands of approximately 25-30 kDa were also detectable. In the partially purified enzyme, the 33-kDa subunit alone was identified as a distinct protein band by SDS-PAGE, and a main basic protein was found at pI 7.6 by native isoelectric focusing. In addition, many bands of more acidic proteins were separated from other enzyme fractions, and protein spots corresponding to the 33 and/or approximately 25-30-kDa subunits were detected in each of the more acidic proteins by two-dimensional gel electrophoresis. CONCLUSION: These results suggest that lambda-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.

Amino Acid Sequence↗
PubMed2006

A comparative study of water distribution, free radical production and activation of antioxidative metabolism in germinating pea seeds.

The aim of this study was to investigate whether there is a relationship between hydration of the embryo axes and cotyledons and the resumption of the oxidative metabolism in both organs of germinating seeds of pea (Pisum sativum L. cv. Piast). Nuclear magnetic resonance ((1)H-NMR) spectroscopy and imaging were used to study temporal and spatial water uptake and distribution in pea seeds. The observations revealed that water penetrates into the seed through the hilum, micropyle and embryo axes, and cotyledons hydrate to different extents. Thus, inhomogeneous water distribution may influence the resumption of oxidative metabolism. Electron paramagnetic resonance (EPR) measurements showed that seed germination was accompanied by the generation of free radicals with g(1) and g(2) values of 2.0032 and 2.0052, respectively. The values of spectroscopic splitting coefficients suggest that they are quinone radicals. The highest content of free radicals was observed in embryo axes immediately after emergence of the radicle. Glutathione content decreased during the entire germination period in both embryo axes and cotyledons. A different profile was observed for ascorbate, with significant increases in embryo axes, coinciding with radicle protrusion. Electrophoretic analysis showed that superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) were present in dry seeds and were activated later during germination, especially in embryo axes. The presence of all antioxidative enzymes as well as low molecular antioxidants in dry seeds allowed the antioxidative machinery to be active as soon as the enzymes were reactivated by seed imbibition. The observed changes in free radical levels, antioxidant contents and enzymatic activities in embryo axes and cotyledons appear to be more closely related to metabolic and developmental processes associated with preparation for germination, and do not correspond directly to the hydration of the tissues.

Antioxidants↗