Showing papers on "Malic enzyme published in 1999"

The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi.

Abstract: The hypothesis is advanced that NADP(+)-malic enzyme (ME; EC 1.1.1.40) is an important activity in regulating the extent of lipid accumulation in filamentous fungi. In Mucor circinelloides, a fungus capable of accumulating only 25% (w/w, dry wt) lipid, even under the most propitious conditions, ME disappears 15-20 h after nitrogen exhaustion, coincident with the cessation of lipid accumulation. In contrast, ME in Mortierella alpina, a fungus capable of accumulating 50% (w/w, dry wt) lipid, remains active for over 60 h after N-exhaustion during which time lipid accumulation continues. No other enzyme activity studied, including the lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, diacyglycerol acyltransferase, ATP: citrate lyase and the NADPH-generating enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP+:isocitrate dehydrogenase, demonstrated any correlation with the accumulation of storage lipid in either fungus. Full activity of ME is restored in Mr. circinelloides within 4 h by adding NH4+ to the cultures, but this is prevented by adding cycloheximide as an inhibitor of protein synthesis. This suggests that the decrease in ME activity occurs due to down-regulation of the ME gene.

Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme

Abstract: DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for 13 strains of lactic acid bacteria, representing Pediococcus, Leuconostoc, Lactobacillus and Oenococcus genera. The sequences were aligned with the corresponding region of mleS in Lactococcus lactis. The phylogenetic distance matrix tree of all mle sequences was compared with the 16S rRNA phylogenetic tree. The analysis showed that the mle fragment evolved more rapidly than the 16S gene and differently. Pediococcus and Lactobacillus species were intermixed in the 16S rRNA tree whereas they were separated in the mle tree. Leuconostoc mesenteroides and Oenococcus oeni were distinct from other species in the 16S rRNA tree, whereas they were intermixed with Lactobacillus species and Lactococcus lactis in the mle tree. The amino acid sequences deduced from partial mle genes were aligned with 22 malic enzyme sequences and the corresponding phylogenetic tree was constructed. Malic and malolactic enzymes were distinct at the phylogenetic level, except for malic enzymes of yeast and Escherichia coli which were nearer the malolactic enzymes than the other malic enzymes. The analysis of conserved sites showed several interesting amino acids specific to either malic enzyme or malolactic enzyme.

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme.

Abstract: The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].

Effect of clofibrate on malic enzyme and leptin mRNAs level in rat brown and white adipose tissue.

Abstract: Two previous studies have reported contradictory results regarding the effect of fibrates treatment on obese (ob) gene expression in rodents. The purpose of the present study was to reinvestigate this issue. We examined the effect of clofibrate (fibrate derivative) administration for 14 days to rats on malic enzyme (as an adequate control of fibrates action) and leptin mRNAs level in the white and brown adipose tissues (WAT and BAT, respectively). The malic enzyme activity and malic enzyme mRNA level in white adipose tissue increased significantly after clofibrate feeding. In brown adipose tissue, the drug treatment resulted in depression of malic enzyme activity and malic enzyme mRNA level. Under the same conditions, leptin mRNA level did not change in these tissues. The results presented in this paper provide further evidence that the clofibrate (activator of peroxisome proliferator activated receptor alpha), feeding is without effect on ob gene expression in rat white and brown adipose tissue. Furthermore, the present study demonstrates that clofibrate causes opposite effects on malic enzyme gene expression in WAT (up-regulation) and BAT (down-regulation).

Catabolism of proline by procyclic culture forms of Trypanosoma congolense

Abstract: The effect of various metabolic inhibitors on the rate of oxygen consumption by procyclic culture forms of Trypanosoma congolense utilizing proline as substrate was investigated. Cyanide inhibited the rate of oxygen consumption by 81.0±6.7%, malonate inhibited the rate by 51.6±1.6% and Antimycin A by 73.1±5.9%. A combination of cyanide and malonate inhibited the rate of oxygen consumption by 84.9±6.7% while a combination of antimycin A and malonate inhibited the rate by 81.6±7.6%. Rotenone had no effect on the rate of respiration except when the intact cells were first permeabilized by digitonin after which rotenone decreased the rate of respiration by 20–30%. Salicylhydroxamate (SHAM) did not have any effect on the rate of oxygen consumption. Enzymes involved in the catabolism of proline with high activities were: proline dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, NADP-linked malic enzyme, alanine aminotransferase and malate dehydrogenase. Activities of 1-pyrroline-5 carboxylate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and NAD-linked malic enzyme were detectable but lower. The end products of proline catabolism were alanine and glutamate. Unlike the case in Trypanosoma brucei brucei aspartate was not detected. Possible pathways of proline catabolism in procyclic culture forms of T. congolense and of electron transfer are proposed.

Citric-acid cycle key enzyme activities during in vitro growth and metacyclogenesis of Leishmania infantum promastigotes.

Abstract: The activities of 5 key regulatory enzymes in most energetic systems, namely citrate synthase (EC 4.1.3.7, CS), NADP-specific isocitrate dehydrogenase (EC 1.1.1.42, ICDH), succinate dehydrogenase (EC 1.3.99.1, SDH), L-malate dehydrogenase (EC 1.1.1.37, MDH), and decarboxylating malic enzyme (EC 1.1.1.40, ME), were measured during the growth and metacyclogenesis of a cutaneous (CL) and a visceral (VL) strain of Leishmania infantum. As occurs with other Leishmania species, infective promastigotes were present along all phases of growth, but their percentages were higher at the early stationary phase for VL and the end of the same phase for CL. High CS and SDH activities were detected in both strains, as compared with other trypanosomatids, bringing more evidence for an actively functional citric-acid cycle in L. infantum. Both strains showed higher levels of CS, ICDH, and MDH and lower SDH and ME activities when more metacyclic promastigotes were present, but in VL these changes paralleled an increase in glucose consumption, whereas in CL these changes coincided with an NH 3 hyperproduction. This suggests that the energy metabolism during L. infantum growth and metacyclogenesis is affected by regulated enzymes that probably respond to changes in the culture medium in the levels of glucose and amino acids.

Glucocorticoid-dependent induction of HMG-CoA reductase and malic enzyme gene expression by polychlorinated biphenyls in rat hepatocytes.

Abstract: Administration of xenobiotics to rats results in hypercholesterolemia and in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and malic enzyme. To investigate the mechanism of the induction of the enzymes by xenobiotics, the effects of xenobiotics on gene expressions for HMG-CoA reductase, malic enzyme, and cytochrome P-450 in rat liver and in cultured hepatocyte were investigated. The treatment of rats with polychlorinated biphenyls (PCB) as a xenobiotic induced mRNAs for HMG-CoA reductase and malic enzyme as well as CYP2B1/2 (cytochrome P-450b/e). Other xenobiotics, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and chloretone, also increased HMG-CoA reductase mRNA. In an investigation of diurnal rhythm of mRNA for HMG-CoA reductase, the induction by PCB was observed in a dark period. Induced expressions of HMG-CoA reductase gene and malic enzyme gene by PCB were observed in primary cultured rat hepatocytes and showed that the action of PCB on the gene expression relating to lipid metabolism was directed on hepatocytes. The induction was observed only in hepatocytes cultured on Engelbreth-Holm-Swarm sarcoma basement membrane gel (EHS-gel), not on type I collagen, which is usually used for monolayer culture of hepatocytes. The induction of CYP2B1/2 gene expression also was observed only in the cells cultured on EHS-gel. The induction of HMG-CoA reductase and malic enzyme by PCB required dexamethasone. However, the addition of dexamethasone per se to medium containing insulin did not show an inductive effect on levels of mRNA for HMG-CoA reductase and malic enzyme. From the data of diurnal variation and hepatocyte culture experiment, HMG-CoA reductase and malic enzyme are considered to be induced by PCB through the so-called “permissive effect” of glucocorticoid.

Insulin Receptor at the Mouse Hepatocyte Nucleus after a Glucose Meal Induces Dephosphorylation of a 30-kDa Transcription Factor and a Concomitant Increase in Malic Enzyme Gene Expression

Abstract: Insulin receptor translocation to the nucleus may represent a mechanism for activation of transcription factors controlling lipogenic gene expression in the mouse hepatocyte. Insulin stimulation was achieved in vivo by oral glucose feeding of mice deprived of food for 24 h. Hepatocytes were fractionated after the glucose meal and nuclei were purified. Insulin receptor levels and phosphorylation state in nuclei were assessed by immunoassay. Insulin receptor significantly increased from basal levels in hepatocyte nuclei within 15 min of the glucose meal. Immunoassay using antiphosphotyrosine indicated that phosphorylation of nuclear insulin receptor increased, whereas phosphorylation of a 30-kDa DNA-binding protein significantly decreased within 15 min of the glucose meal. Glucose treatment significantly increased expression of malic enzyme within the time frame of insulin receptor translocation to the nucleus. Nuclear protein binding to an insulin response element (IRE) within the malic enzyme gene promoter significantly increased within 15 min of the glucose meal. When cell nuclei were isolated from mice that had been deprived of food and treated in vitro with purified, activated insulin receptor, changes were observed in DNA-binding protein phosphorylation and IRE-binding in the absence of cytoplasmic insulin signaling. In vitro incubation of nuclei with activated insulin receptor significantly decreased phosphorylation of a 30-kDa DNA-binding protein compared with basal levels. Increased binding of nuclear proteins to malic enzyme IRE was observed upon stimulation of isolated nuclei with activated insulin receptor. These results suggest that nuclear insulin receptors induce malic enzyme gene expression by regulating phosphorylation of IRE transcription factors.

Methods for modulating water-use efficiency or productivity in a plant by transforming with a DNA encoding a NAPD-malic enzyme operably linked to a guard cell or an epidermal cell promoter

Abstract: A method for modulating water-use efficiency or productivity in a plant is provided. In particular, stomatal aperture in a plant can be regulated by transforming the plant with a polynucleotide that encodes a NADP-malic enzyme operably linked to a guard cell or an epidermal cell promoter that modifies malate accumulation in the stomata.

New nucleic acid encoding malic enzyme, useful for increasing yield of L-lysine in fermentation of transformed cells

Abstract: Nucleic acid (I) having a sequence at least 50% identical with the gene encoding the malic enzyme (ME) of Corynebacterium glutamicum, is new. Independent claims are also included for the following: (a) polypeptide (II) with a sequence at least 75% identical with that of ME of C. glutamicum; (b) expression vector comprising a sequence (Ia) encoding an ME fused to a promoter functional in prokaryotic and/or eukaryotic cells, preferably C. glutamicum; (c) cells transformed with the vector of (b); and (d) production of L-lysine in cells that overexpress ME.

Effect of hexachlorobenzene on NADPH-generating lipogenic enzymes and L-glycerol-3-phosphate dehydrogenase in brown adipose tissue.

Abstract: The effect of the in vivo administration of hexachlorobenzene (HCB) (100 mg/100 g bw) for 30 days, on the activities of brown adipose tissue (BAT) lipogi.e. malic enzyme (ME), and glucose-6-enic enzymes, phosphate dehydrogenase (G6PD) and the mitochondrial non lipogenic enzyme, L-glycerol-3-phosphate dehydrogenase (LG3PD), was studied in male Wistar rats, submitted to various neurohormonal manipulations. BAT ME, G6PD and LG3PD activities showed significant reductions in response to HCB treatment both in euthyroid and surgically thyroidectomized rats, showing that the effect does not depend on the presence of thyroid hormones. These results differ from those obtained for hepatic ME and G6PD activities, which increased in HCB intoxicated rats without alteration in LG3PD. HCB decreased BAT ME activity under BAT denervation. Administration of HCB resulted in time and dosedependent decreases in the activity of BAT malic enzyme. The basal activity of ME was increased in hypothyroid rats, while that of LG3PD was reduced. A stimulatory effect of receptor-saturating doses of triiodothyronine (T3) (50 μg/100 g body weight) was observed on BAT ME and LG3PD activities in thyroidectomized rats, showing that the enzymes responded to thyroid hormone stimulation in a normal manner. The stimulatory effect of saturating doses of T3 on ME and LG3PD was reduced by HCB. The results presented herein unequivocally show that brown adipose tissue is a specific target in HCB-induced toxicity, which in turn involves severe alterations in the regulation of BAT lipogenesis.

Crystallization and preliminary x-ray diffraction analysis of malic enzyme from pigeon liver.

Abstract: Recombinant pigeon-liver malic enzyme was expressed in Escherichia coli and purified to homogeneity. Two different crystal forms were grown by the hanging-drop vapour-diffusion method. Both types of crystals belong to the tetragonal space group P4222, with unit-cell dimensions a = b = 163.8, c = 174.3 A for the octahedral crystals and a = b = 124.5, c = 179.2 A for the rod-like crystals. X-ray diffraction data were collected at 100 K using a synchrotron-radiation X-ray source. The Matthews parameter suggests that there are four and two molecules per asymmetric unit for the larger and the smaller tetragonal unit cells, respectively.

Potentiometric Determination of L - Malate Using Ion - Selective Electrode in Flow Injection Analysis System

Abstract: A potentiometric biosensor employing a CO₃^(2-) ion-selective electrode (ISE) and malic enzyme immobilization in a flow injection analysis (FIA) system was constructed. Analytical parameters were optimized for L-malate determination. The CO₃^(2-)-ISE-FIA system was composed of a pump, an injector, a malic enzyme (EC 1.1.1.40) reactor, a CO₃^(2-) ion-selective electrode, a pH/㎷ meter and a recorder. Cofactor NADP was also injected with substrate for the enzyme reaction into the system. Optimized analytical parameters for L-malate determination in the CO₃^(2-)-ISE-FIA system were as follows: flow rate, 14.5 ml/hr; sample injection volume, 100 μl; enzyme loading in the reactor, 20 units; length of the enzyme reactor, 7 ㎝; tubing length from the enzyme reactor to the detector as a geometric factor in FIA, 15 ㎝. The response time for measuring the entire L-malate concentration range 10^(-2)~10^(-5) mol/L; 4 injections) was <15 minutes. In this CO₃^(2-)-ISE-FIA system, the potential differences due to the formation of CO₃^(2-) by the reaction of malic enzyme on L-malate were correlated to L-malate concentration in the range of 10^(-2)~10^(-5) mol/L; the detection limit was 10^(-5) mol/L. This potentiometric CO₃^(2-)-ISE-FIA system was found to be useful for L-malate measurement.

Kinetic behavior and protein expression of hepatic NADPH-production systems during development of

Abstract: We have studied the evolution of the kinetic behavior of main NADPH-generating systems, . . glucose-6-phosphate dehydrogenase G6PDH , 6-phosphogluconate dehydrogenase 6PGDH , . . malic enzyme ME and isocitrate dehydogenase NADP-dependent NADP-IDH in liver of rainbow trout at different developmental stages. Our results show that during development, liver growth was due to an increase in the number of cells. Under these conditions, the main kinetic parameters and pattern of protein expression of these enzyme systems did not change in any of the developmental stages studied. However, when the values of these kinetic parameters were expressed in terms of cell units, we found progressive and significant decreases in all enzymes assayed as fish size increased, indicating that during development, the liver makes less total NADPH available to the cell. q 1999 Elsevier Science B.V. All rights reserved.