Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles

In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and alpha-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in alpha-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of alpha-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of alpha-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.     

Effect of Calcium and Zinc on Subcellular Distribution, Activity and Thermosensitivity of Superoxide Dismutase in Mnium Affine

In the leafstem moss Mnium affine two superoxide dismutase (SOD) isoforms were found in chloroplasts and two in mitochondria. Four other isozymes were probably cytosolic and two of them had high activity and thermostability and were very sensitive to H₂O₂. On the other hand, one of the mitochondrial isoenzymes was very sensitive to high temperature. The activity and thermosensitivity of SOD was considerably dependent on calcium and zinc ions. The effect was different for the individual isoforms and related to their subcellular distribution. Calcium ions predominantly activated and stabilized one cytosolic and the mitochondrial SODs, while zinc ions influenced one chloroplastic and two cytosolic SODs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Subcellular distribution of serine hydroxymethyltransferase in Plasmodium lophurae.

Subcellular fractions of the bird malaria, $\text{Plasmodium lophurae}$ were prepared by differential centrifugation. Cytochrome oxidase activity was located in the mitochondrial fraction. A major portion of glutamate dehydrogenase activity was found in the mitochondrial fraction with the remainder in the ribosomal and cytosolic fractions. Malate dehydrogenase and serine hydroxymethyltransferase activities were located primarily in the cytosolic fraction.     

An NADP-Glutamate Dehydrogenase from the Green Alga Bryopsis maxima. Purification and Properties

NADP-glutamate dehydrogenase (EC 1.4.1.4 [EC] ; NADP-GDH) was purifiedto electrophoretic homogeneity from the multinuclear-unicellulargreen marine alga in Sipho-nales, Bryopsis maxima, and its propertieswere examined. M_r of the undenatured enzyme was 280 kDa, andthe enzyme is thought to be a hexamer of 46 kDa subunit protein.Optimum pHs for the reductive amination and oxidative deaminationwere 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specificactivities with a ratio of 18 :1. Apparent K_m values for 2-oxoglutarate,ammonia, NADPH, glutamate and NADP⁺ were 3.0, 2.2, 0.03, 3.2and 0.01 mM respectively. The enzymochemical characteristicsof the GDH were studied and compared to those of other species.The B. maxima GDH was insensitive to 5 mM Ca²⁺ and to 1 mM EDTAin contrast to higher plant NAD-GDHs. Chemical modificationswith DTNB and pCMBS suggested that cysteine residues are essentialfor the enzymatic activity as in other species GDHs. The GDHwas not affected by 1 mM purine nucleotides, suggesting thatthe enzyme is not allosteric, in contrast to animal NAD(P)-GDHsand fungal NAD-GDHs. (Received August 12, 1996; Accepted January 7, 1997)     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH     

Induction of new isoforms of superoxide dismutase and catalase enzymes in the flag leaf of wheat during monocarpic senescence.

Leaf senescence is a programmed cell death phenomenon and involves oxidative stress. Superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT EC 1.11.1.6) activities were studied in the flag leaf of Triticum aestivum cv. Kundan at different stages of grain development. Both SOD and CAT activities showed a decline during monocarpic senescence. Three SOD isozymes were observed in the cytosol, of which one isozyme was observed in the chloroplasts as well. Mitochondria showed the presence of three low abundant SOD isoforms. Inhibitor studies revealed the cytosolic and chloroplastic isoforms to be Cu/Zn SODs. In mitochondria however, two isozymes were MnSOD and one of them appeared to be FeSOD. These isoforms present in the mitochondria increased in activity as senescence progressed. Three isoforms of CAT were observed in peroxisomes which responded differentially during monocarpic senescence. The changes in the kind and pattern of the antioxidant enzymes supported the ordered sequence of events during leaf senescence. This is the first report showing an increase in mitochondrial FeSOD activity during leaf senescence.     

Involvement of GDH3-encoded NADP+-dependent Glutamate Dehydrogenase in Yeast Cell Resistance to Stress-induced Apoptosis in Stationary Phase Cells

Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP⁺-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.     

Evolutionary analysis of aspartate aminotransferases

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family. Correspondence to: C.J. Marshall     

Mutants of Pseudomonas aeruginosa unable to inactivate allantoinase and NADP-dependent glutamate dehydrogenase

Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.Abbreviations NADP-GDH NADP-dependent glutamate dehydrogenase - Ani^- mutant allantoinase non-inactivating mutant - GOGAT glutamate synthase     

Both chloroplastic and cytosolic phosphoglycerate kinase isozymes are present in the pea leaf nucleus

Summary. Phosphoglycerate kinase (EC 2.7.2.3) occurs in chloroplasts, cytosol, and nuclei in higher plants. Immunocytolocalization experiments with isozyme-specific antibodies indicate that both the chloroplastic and the cytosolic forms of the enzyme are present in the pea (Pisum sativum L.) leaf nucleus.Correspondence and reprints: Department of Biological Sciences m/c 066, University of Illinois–Chicago, 845 West Taylor, Chicago, IL 60607-7060, U.S.A.     

Compartmentation and Activities of Enzymes Involved in the Metabolism of Amino Acids Implicated in Osmoregulatory Mechanisms in Acanthamoeba castellanii1

Glutamate dehydrogenase in Acanthamoeba castellanii is an NAD-dependent cytosolic enzyme. This is similar to glutamate dehydrogenases in Phycomycetes, but very different from the dual coenzyme-specific enzymes located in mitochondria in animals and in mitochondria and chloroplasts in higher plants. Pyrroline-5-carboxylate (P-5-C) reductase occurs also in the cytoplasm in A. castellanii and has very high affinities for L-P-5-C (K_m= 12 μM) and NADH (K_m= 15 _μM). In contrast, ornithine aminotransferase and proline oxidase are mitochondrial enzymes. No proline-inhibited γ-glutamyl kinase was detected while an active glutamine synthetase was found in the cytosolic compartment. Evidence for a mitochondrial transport system for L-proline was obtained. Two possible pathways for proline biosynthesis in A. castellanii are discussed based on information obtained about activities and subcellular compartmentation of enzymes.     

Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae

The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described. Two DNA fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh- strain of S. cerevisiae and directs substantial overproduction of NADP-GDH. One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N. crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.     

Cadmium accumulation in the chloroplast of Euglena gracilis

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 m M CdCl₂, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd²⁺, since uptake of added CdCl₂ in isolated chloroplasts exhibited a hyperbolic type of kinetics with a K_m of 57 µ M and V_max of 3.7 nmol (mg protein)⁻¹ min⁻¹. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd²⁺, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.     

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.     

Regulation of nitrogen-metabolizing enzymes in the commercial Mushroom Agaricus bisporus

Mycelium of Agaricus bisporus strain Horst U1 was grown in batch cultures on different concentrations of ammonium, glutamate, and glucose to test the effect of these substrates on the activities of NADP-dependent glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4), NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2.), and glutamine synthetase (GS, EC 6.3.1.2.). When grown on ammonium, the activities of NADP-GDH and GS were repressed. NAD-GDH activity was about 10 times higher than the activities of NADP-GDH and GS. At concentrations below 8 mM ammonium, NADP-GDH and GS were slightly derepressed. When glutamate was used as the nitrogen source, activities of NADP-GDH and GS were derepressed; compared with growth on ammonium, the activities of these two enzymes were about 10 times higher. Activities of GDHs showed no variation at different glutamate concentrations. Activity of GS was slightly derepressed at low glutamate concentrations. Growth of A. bisporus on both ammonium and glutamate as nitrogen sources resulted in enzyme activities comparable to growth on ammonium alone. Activities of NADP-GDH, NAD-GDH, and GS were not influenced by the concentration of glucose in the medium. In mycelium starved for nitrogen, the activities of NADP-GDH, NAD-GDH, and GS were derepressed, while in carbon-starved mycelium the activity of GS and both GDHs was repressed.     

Adenylate Levels, Energy Charge, and Phosphorylation Potential during Dark-Light and Light-Dark Transition in Chloroplasts, Mitochondria, and Cytosol of Mesophyll Protoplasts from Avena sativa L

The compartmentation of cellular energy relations during dark-light and light-dark transitions was studied by means of a newly developed technique to fractionate oat (Avena sativa L., var. Arnold) mesophyll protoplasts. Using an improved microgradient system with hydrophobic and hydrophilic layers of increasing density, a pure plastid pellet (up to 90% of total chloroplasts) could be separated from an interphase of only slightly contaminated mitochondria (70 to 80% of total mitochondria), and a cytoplasmic supernatant could be obtained within 60 seconds. Appropriate controls indicate that, under the conditions employed, metabolic interconversions of adenylates can be kept to a minimum and, thus, be determined and corrected for. Cross contamination of the fractions, as well as liberation of organelles to the supernatant, was assessed by specific markers, and the metabolite levels recorded were corrected accordingly. Using this technique, we found that, during dark-light transition, the chloroplastic and cytosolic ATP exhibits a rapid increase, while the mitochondrial ATP level decreases. In all compartments, ADP levels mirror alterations of the ATP pool in the opposite way, at least to some extent. To compensate fully for the rise in ATP, chloroplastic and mitochondrial AMP levels change accordingly, indicating that, due to the more or less unchanged level of total adenylates, there is no net flux of adenylates between the compartments. In contrast to the organelles, no AMP could be detected within the cytosol. When the light is turned off, a decrease of ATP coincides between chloroplast stroma and the cytosol for only about 30 seconds. Under prolonged dark treatment, cytosolic ATP rises again, while stroma ATP levels exhibit a further decrease. After about 60 seconds of darkness, the cytosolic ATP level is back to its initial value. This obviously is due to the immediate rise in mitochondrial ATP upon darkening, which cumulates after about 60 seconds; then, caused by an ATP/ADP exchange with the cytosol, it levels off again at the state before changing the conditions, as soon as the cytosolic ATP is also back to its original level. All of these events are closely mirrored by the change in the ATP/ADP ratio and the energy charge within the compartments. While the values for chloroplasts exhibit considerable differences between dark and light, those calculated for mitochondria and the cytosol exhibit only transient changes. These are limited to about 60 seconds of undershoot or overshoot, with respect to the cytosol, and then return to nearly the levels observed before changing the conditions. Adenylate kinase was found to be exclusively associated with chloroplasts (90% of total activity level) and mitochondria. Isotonic liberation of vacuoles did not point toward a significant association of adenylates with this compartment.     

Partial purification and characterization of 3-hydroxy-3-methylglutaryl coenzyme a reductase from the leaves of guayule (Parthenium argentatum)

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been isolated and was partially purified from the leaves of Parthenium argentatum. The enzyme was found to be associated both with the cytosol and the chloroplasts. Ten mM dithiothreitol was essential to prevent loss of activity. Optimum activities of cytosolic and chloroplastic fractions were observed at pH 7.0 and 7.5 respectively. Preincubation of the reaction mixtures with CoA, acetyl-CoA, σ-phenanthroline and iodoacetamide resulted in the progressive loss of enzyme activity. 3-Hydroxybutyrate and mevalonate also inhibited the enzyme. The Michaelis constants of the enzyme for HMG-CoA and NADPH were 0.25 and 0.31 mM respectively for the cytosolic enzyme, while those for the chloroplastic enzyme were 0.018 and 0.42 mM respectively. Inhibition studies indicated that hydroxybutyrate was a competitive inhibitor with respect to HMG-CoA. The inhibition of mevalonate was competitive with HMG-CoA and non-competitive with NADPH.