Malate valves: old shuttles with new perspectives.

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyse the reversible interconversion of malate and oxaloacetate and their transport. Depending on the co-enzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes. Activities of NAD-dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids. In addition, chloroplasts possess a NADP-dependent MDH isoform. The NADP-MDH as part of the 'light malate valve' plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post-translational redox-modification mediated via the ferredoxin-thioredoxin system and fine control via the NADP+ /NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD-MDH ('dark malate valve') is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, knowledge about regulation of the other two cytosolic MDHs as well as NAD-MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria and peroxisomes have been characterised, but not much is known about cytosolic NAD-MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange, focusing on the various metabolic functions of these valves.

Emerging concept for the role of photorespiration as an important part of abiotic stress response.

When plants are exposed to stress, generation of reactive oxygen species (ROS) is often one of the first responses. In order to survive, cells attempt to down-regulate the production of ROS, while at the same time scavenging ROS. Photorespiration is now appreciated as an important part of stress responses in green tissues for preventing ROS accumulation. Photorespiratory reactions can dissipate excess reducing equivalents and energy either directly (using ATP, NAD(P)H and reduced ferredoxin) or indirectly (e.g., via alternative oxidase (AOX) and providing an internal CO2 pool). Photorespiration, however, is also a source of H2 O2 that is possibly involved in signal transduction, resulting in modulation of gene expression. We propose that photorespiration can assume a major role in the readjustment of redox homeostasis. Protection of photosynthesis from photoinhibition through photorespiration is well known. Photorespiration can mitigate oxidative stress under conditions of drought/water stress, salinity, low CO2 and chilling. Adjustments to even mild disturbances in redox status, caused by a deficiency in ascorbate, AOX or chloroplastic NADP-malate dehydrogenase, comprise increases in photorespiratory components such as catalase, P-protein of glycine decarboxylase complex (GDC) and glycine content. The accumulation of excess reducing equivalents or ROS in plant cells also affects mitochondria. Therefore, a strong interaction between the chloroplast redox status and photorespiration is not surprising, but highlights interesting properties evident in plant cells. We draw attention to the fact that a complex network of multiple and dynamic systems, including photorespiration, prevents oxidative damage while optimising photosynthesis. Further experiments are necessary to identify and validate the direct targets of redox signals among photorespiratory components.

Glutamine and alanyl-glutamine dipeptide reduce mesenteric plasma extravasation, leukocyte adhesion and tumor necrosis factor-α (TNF-α) release during experimental endotoxemia.

Glutamine (GLN) appears to be an essential nutrient during organism development and critical illness. The aim of our study was to evaluate the effects of GLN and its generic preparation alanyl-glutamine-dipeptide (DIP) on the microcirculation in endotoxemia in rats and its effects on tonus or aortal rings in vitro. Male Lewis rats (n=40) were separated in 4 groups. Group 1 (CON) served as healthy control group while the other groups received an endotoxin bolus i.v. (5 mg/kg lipopolysaccharide, LPS i.v.). In group 3 (LPS+GLN) 0.75 g/kg-1 GLN i.v. before LPS challenge was administered. In group 4 (LPS+DIP) DIP containing 0.75 g/kg GLN was given. Leukocyte-endothelial interactions and mesenteric plasma extravasation were determined at 0, 1 and 2 hours during the experiment by intravital fluorescence microscopy (IVM). Cytokine release (TNF-alpha, IL-1 beta, IL-6, IL-10) was measured by ELISA. GLN treatment reduced leukocyte adherence (-49.7% vs. LPS group, p<0.05) and plasma extravasation (-12.3% vs. LPS group, p<0.05) significantly during endotoxemia compared to untreated LPS animals. In group 4 (DIP+LPS), a decrease of leukocyte adherence (-56.0%) and mesenteric plasma extravasation (-18.8% vs. LPS group, p<0.05) was also found. TNF-alpha levels were reduced in both GLN and DIP (p<0.05). In vitro experiments demonstrated that glutamine agents could attenuate the response to contracting agents in presence of the vascular endothelium, implying nitric oxide pathway. In vivo, GLN as well as DIP pre-treatment diminish the detrimental impact of endotoxemia on the mesenteric microcirculation and the TNF-alpha release, the effects whose clinical importance should be further examined.

How well can epileptic seizures be predicted? An evaluation of a nonlinear method.

The unpredictability of the occurrence of epileptic seizures contributes to the burden of the disease to a major degree. Thus, various methods have been proposed to predict the onset of seizures based on EEG recordings. A nonlinear feature motivated by the correlation dimension is a seemingly promising approach. In a previous study this method was reported to identify 'preictal dimension drops' up to 19 min before seizure onset, exceeding the variability of interictal data sets of 30-50 min duration. Here we have investigated the sensitivity and specificity of this method based on invasive long-term recordings from 21 patients with medically intractable partial epilepsies, who underwent invasive pre-surgical monitoring. The evaluation of interictal 24-h recordings comprising the sleep-wake cycle showed that only one out of 88 seizures was preceded by a significant preictal dimension drop. In a second analysis, the relation between dimension drops within time windows of up to 50 min before seizure onset and interictal periods was investigated. For false-prediction rates below 0.1/h, the sensitivity ranged from 8.3 to 38.3% depending on the prediction window length. Overall, the mean length and amplitude of dimension drops showed no significant differences between interictal and preictal data sets.

The seizure prediction characteristic: a general framework to assess and compare seizure prediction methods.

The unpredictability of seizures is a central problem for all patients suffering from uncontrolled epilepsy. Recently, numerous methods have been suggested that claim to predict from the EEG the onset of epileptic seizures. In parallel, new therapeutic devices are in development that could control upcoming seizures provided that their onset is known in advance. A reliable clinical application controlling seizures, consisting of a seizure prediction method and an intervention system, would improve patient quality of life. The question therefore arises as to whether the performance of the seizure prediction methods is already sufficient for clinical applications. The answer requires assessment criteria to judge and compare these methods, but recognized criteria still do not exist. Based on clinical, behavioral, and statistical considerations, we suggest the "seizure prediction characteristic" to evaluate seizure prediction methods. Results of this approach are exemplified by its application to the "dynamical similarity index" seizure prediction method using 582 hours of intracranial EEG data, including 88 seizures.

Reset of human neocortical oscillations during a working memory task.

Both amplitude and phase of rhythmic slow-wave electroencephalographic activity are physiological correlates of learning and memory in rodents. In humans, oscillatory amplitude has been shown to correlate with memory; however, the role of oscillatory phase in human memory is unknown. We recorded intracranial electroencephalogram from human cortical and hippocampal areas while subjects performed a short-term recognition memory task. On each trial, a series of four list items was presented followed by a memory probe. We found agreement across trials of the phase of oscillations in the 7- to 16-Hz range after randomly timed stimulus events, evidence that these events either caused a phase shift in the underlying oscillation or initiated a new oscillation. Phase locking in this frequency range was not generally associated with increased poststimulus power, suggesting that stimulus events reset the phase of ongoing oscillations. Different stimulus classes selectively modulated this phase reset effect, with topographically distinct sets of recording sites exhibiting preferential reset to either probe items or to list items. These findings implicate the reset of brain oscillations in human working memory.

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture.

The addition of cyclosporin A (500 ng ml(-1)) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 x 10(-7) M) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.

A soybean plastid-targeted NADH-malate dehydrogenase: cloning and expression analyses.

A typical soybean (Glycine max) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2-ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyruvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule-enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana, which has a completely different transit peptide. In addition, the soybean nodule-enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of ∼34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene.

Light-modulated NADP-malate dehydrogenases from mossfern and green algae: insights into evolution of the enzyme's regulation.

Chloroplast NADP-dependent malate dehydrogenase is one of the best-studied light-regulated enzymes. In C3 plants, NADP-MDH is a part of the 'malate valve' that controls the export of reducing equivalents in the form of malate to the cytosol. NADP-MDH is completely inactive in the dark and is activated in the light with reduced thioredoxin. Compared with its permanently active NAD-linked counterparts, NADP-MDH exhibits N- and C-terminal sequence extensions, each bearing one regulatory disulphide. Upon reduction of the C-terminal disulphide, the enzyme active site becomes accessible for the substrate. Reduction of the N-terminal disulphide promotes a conformational change advantageous for catalysis. To trace the evolutionary development of this intricate regulation mechanism, we isolated cDNA clones for NADP-MDH from the mossfern Selaginella and from two unicellular green algae. While the NADP-MDH sequence from Selaginella demonstrates the classic cysteine pattern of the higher plant enzyme, the sequences from the green algae are devoid of the N-terminal regulatory disulphide. Phylogenetic analysis of new sequences and of those available in the databases led to the conclusion that the chloroplast NADP-MDH and the cytosolic NAD-dependent form arose via duplication of an ancestral eubacterial gene, which preceded the separation of plant and animal lineages. Redox-sensitive NADP-MDH activity was detected only in the 'green' plant lineage starting from the primitive prasinophytic algae but not in cyanobacteria, Cyanophora paradoxa, red algae and diatoms. The latter organisms therefore appear to utilize mechanisms other than the light-regulated 'malate valve' to remove from plastids excessive electrons produced by photosynthesis.

Functional coupling of cyclooxygenase 1 and 2 to discrete prostanoid synthases in liver macrophages.

The profile of released prostanoids after addition of exogenous arachidonic acid to resident liver macrophages is different from the profile obtained in lipopolysaccharide-pretreated cells. In resident and lipopolysaccharide-pretreated cells, AA leads to a release of thromboxane B(2), prostaglandin F(2alpha), E(2), and D(2). A specifically enhanced formation of prostaglandin E(2) is obtained in lipopolysaccharide-pretreated cells. Resident liver macrophages express cyclooxygenase 1, and thromboxane A(2)-, prostaglandin F(2alpha)-, E(2)-, and D(2)-synthase. Treatment with lipopolysaccharide induces-in addition to cyclooxygenase 2-an enhanced expression of the prostaglandin E(2) synthase. In resident liver macrophages, the formation of prostanoids from exogenous arachidonic acid is completely inhibited by SC560 (a specific inhibitor of cyclooxygenase 1), but remains unchanged with SC236 (a specific inhibitor of cyclooxygenase 2). In lipopolysaccharide-pretreated liver macrophages, the formation of thromboxane B(2), prostaglandin F(2alpha) and D(2) is equally inhibited by SC560 and SC236 by about 50%. In contrast, the formation of prostaglandin E(2) is inhibited to a greater extent by SC560 (75%) compared to SC236 (26%). We conclude from these data, that in lipopolysaccharide-pretreated liver macrophages (i) cyclooxygenase 1 and 2 couple both to discrete prostanoid synthases, (ii) the functional coupling of cyclooxygenase 1 and 2 to the thromboxane A(2)-, prostaglandin F(2alpha)-, and D(2)-synthase is almost identical, and (iii) the enhanced prostaglandin E(2) synthesis is due to an enhanced expression of the prostaglandin E(2) synthase, which is coupled more efficiently to cyclooxygenase 1.

Reversible Ca2+-induced fast-to-slow transition in primary skeletal muscle culture cells at the mRNA level.

1. The adult fast character and a Ca2+-inducible reversible transition from a fast to a slow type of rabbit myotube in a primary culture were demonstrated at the mRNA level by Northern blot analysis with probes specific for different myosin heavy chain (MyHC) isoforms and enzymes of energy metabolism. 2. No non-adult MyHC isoform mRNA was detected after 22 days of culture. After 4 weeks of culture the fast MyHCIId mRNA was strongly expressed while MyHCI mRNA was virtually absent, indicating the fast adult character of the myotubes in the primary skeletal muscle culture. 3. The data show that a fast-to-slow transition occurred in the myotubes at the level of MyHC isoform gene expression after treatment with the Ca2+ ionophore A23187. The effects of ionophore treatment were decreased levels of fast MyHCII mRNA and an augmented expression of the slow MyHCI gene. Changes in gene expression started very rapidly 1 day after the onset of ionophore treatment. 4. Levels of citrate synthase mRNA increased and levels of glyceraldehyde 3-phosphate dehydrogenase mRNA decreased during ionophore treatment. This points to a shift from anaerobic to oxidative energy metabolism in the primary skeletal muscle culture cells at the level of gene expression. 5. Withdrawal of the Ca2+ ionophore led to a return to increased levels of MyHCII mRNA and decreased levels of MyHCI mRNA, indicating a slow-to-fast transition in the myotubes and the reversibility of the effect of ionophore on MyHC isoform gene expression.

Regulation in metabolic systems under homeostatic flux control.

The general properties of metabolic systems under homeostatic flux control are analyzed. It is shown that the main characteristic point for an enzyme in such a system is a sharp transition from limitation outside the system to limitation by some enzyme inside the system. A method for the quantitative treatment of the experimental dependence of metabolic flux on enzyme content is presented. The conception of "nonlimiting," "near-limiting," and "limiting" enzymes is developed for these systems. It is pointed out that reactions close to a thermodynamic equilibrium under normal conditions can considerably limit the homeostatic fluxes. The rules for regulation of fluxes in such systems are illustrated by the data obtained for transgenic plants with reduced activities of some Calvin-cycle enzymes and further examples. A comparison is made between the developed quantitative description of metabolic fluxes under homeostatic flux control and the methods of metabolic control analysis.

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid.

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.

Electron acceptors in isolated intact spinach chloroplasts act hierarchically to prevent over-reduction and competition for electrons.

Electron fluxes in isolated intact spinach chloroplasts were analyzed under saturating light and under optimal CO(2) and P(i) supply. When CO(2) assimilation was the only ATP- and NADPH-consuming reaction, the DeltapH decreased and the chloroplasts showed clear evidence of over-reduction. This suggested that additional electron flow is required in order to maintain the DeltapH and the stromal NADPH/ATP ratio. The additional electron flow may be cyclic electron transport around Photosystem I and linear electron transport towards either oxaloacetate or O(2). The contributions of, and the interrelationships between, these three electron transfer pathways were analyzed by following the reactions of chloroplasts in their presence or absence, and by monitoring to what extent they were able to compensate for each other. Inhibition of cyclic electron flow by antimycin A caused strong over-reduction and decreased the DeltapH. Only oxaloacetate, but not O(2), was able to restore photosynthesis. In the presence of H(2)O(2), there was a rapid build-up of a high DeltapH, and the reduction of any other electron acceptor was prevented. It is concluded that the different electron acceptors in the stroma are organized in a hierarchical manner; this allows electron flux towards CO(2) and nitrite reduction to proceed without any competition for electrons, and any excess electrons to be taken by these additional non-assimilatory pathways. Hence, the DeltapH is maintained at the required level and over-reduction of the electron transport chain and the stromal redox components is avoided.

Effects of retinoic acid on N-glycosylation and mRNA stability of the liver/bone/kidney alkaline phosphatase in neuronal cells.

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N-linked glycosylation of the tissue nonspecific liver/bone/kidney- type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N-glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 microgram/ml) of tunicamycin for 48 h, uninduced and RA-induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N-linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N-glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [(14)C]mannose and [(35)S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [(14)C]mannose incorporation into ALP molecules. Also, the release of free [(14)C]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N-glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further.

Regulation of the Calvin cycle for CO2 fixation as an example for general control mechanisms in metabolic cycles.

The theory of a metabolic cycle with the main portion of its intermediates remaining inside the cycle during one turnover has been developed. On this basis, the regulation of the Calvin cycle is analyzed. It is demonstrated that not only the reactions of non-equilibrium enzymes, as the carboxylation of ribulose 1,5-bisphosphate, but reactions that operate close to a thermodynamic equilibrium, especially the reduction of 3-phosphoglycerate and the transketolase reaction can significantly influence the total turnover period in the Calvin cycle. The role of compensating mechanisms in the maintenance of the photosynthesis rate upon changes of environmental conditions and of enzyme contents is analyzed for the Calvin cycle. It is shown that the change of the total quantity of the metabolites is one of the main self-regulated mechanisms in the Calvin cycle. A change of the ATP/ADP ratio can be used by the cell to maintain the CO2 assimilation rate, when the total quantity of the metabolites is changed. The developed analysis permits to explain some experimental data obtained with transgenic plants with restricted efflux of carbon from the chloroplasts.

Regulation of UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase by retinoic acid in P19 cells.

UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase (GPT) is the first enzyme in the dolichol pathway of protein N-glycosylation, and is implicated in the developmental programmes of a variety of eukaryotes. In the present study we describe the effects of all-trans-retinoic acid (RA) on the levels of GPT protein and enzymic activity, and on the transcription rate of the GPT gene, in mouse P19 teratocarcinoma cells. RA caused a dose-dependent and protein-synthesis-dependent induction of enzyme activity. The maximum induction of GPT activity (about 3-fold) required 2 days of exposure to 1 microM RA. Induced GPT activity also resulted in an increase in the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2. Enzymic activities paralleled GPT gene expression. The GPT gene was induced (2-fold) after 7 h of RA treatment. An approx. 3-fold increase in a 48 kDa GPT protein and approx. 4-fold increases in the levels of three GPT transcripts (1.8, 2.0 and 2.2 kb) were observed after 2 days of RA treatment. The enhanced levels of GPT protein and mRNAs began to decline 3 days after the initiation of differentiation, and GPT expression was down-regulated during cellular differentiation. GPT activity decreased about 2. 8-fold to a constant level in differentiated P19 cells. The results indicate that the RA-induced enzyme activity was mainly determined by increased transcription of the GPT gene. RA-treated P19 cells were about 4-fold more resistant to tunicamycin, a fungal antibiotic which inhibits GPT, than were control cells. In addition, GPT activity in membranes from RA-treated P19 cells exhibited approx. 4-fold increased resistance to tunicamycin compared with activity in membranes from untreated control cells, demonstrating that resistance to tunicamycin is correlated with induced GPT activity. Furthermore, increased GPT activity had regulatory significance with regard to the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and into glycoproteins. Together, the data provide additional insights into the hormonal regulation of GPT and present evidence that the RA-mediated induction of GPT has a regulatory impact on the dolichol pathway.

A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L.

We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1. 1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC 1.1.1.82). Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated from Arabidopsis and spinach. A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase. A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa. Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts. Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues. The findings reported here indicate that the "malate valve" operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids.

Flux control of the malate valve in leaf cells.

The coupled processes of the chloroplast trans-envelope transport of malate and oxaloacetate and their interconversion as catalyzed by the stromal NADP-linked malate dehydrogenase are quantitatively analyzed by means of a steady-state model. The equation for the NADP-malate dehydrogenase reaction is developed. The empirical dependence of enzyme activity on NADPH and NADP+ is used to determine its actual activity. The trans-envelope counter exchange of malate and oxaloacetate is described by a kinetic model of the translocator. Kinetic parameters are derived from known data, except for the Km value and the maximum rate for oxaloacetate transport, which are estimated from oxaloacetate-dependent malate formation in isolated intact chloroplasts. Using the kinetic properties of the system and the known metabolite concentrations, the model demonstrates that photosynthetically generated NADPH can be exported efficiently from the chloroplasts to the cytosol by the malate-valve system. The transfer capacity of the malate valve is estimated not to exceed 20 mumol (mg Chl)-1 h-1 (or 5% of the electron transport) under normal physiological conditions. The possible role of the malate valve in leaf cells under normal conditions and during stress is discussed.

Cloning and sequence analysis of cDNAs encoding plant cytosolic malate dehydrogenase.

Here we report the first complete sequence of plant cytosolic malate dehydrogenase (EC 1.1.1.37). The phylogenetic relationships between malate dehydrogenases from different cell compartments are discussed. The constructed phylogenetic tree shows that cytosolic NAD-MDH and chloroplast NADP-MDH have evolved through gene duplication of the pre-existing nuclear gene.