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PubMed1997

Expression of Ascaris suum malic enzyme in a mutant Escherichia coli allows production of succinic acid from glucose.

The malic enzyme gene of Ascaris suum, was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

Animals↗
PubMed2005

Chronic intermittent hypoxia upregulates genes of lipid biosynthesis in obese mice.

Obstructive sleep apnea (OSA), a condition tightly linked to obesity, leads to chronic intermittent hypoxia (CIH) during sleep. There is emerging evidence that OSA is independently associated with insulin resistance and fatty liver disease, suggesting that OSA may affect hepatic lipid metabolism. To test this hypothesis, leptin-deficient obese (ob/ob) mice were exposed to CIH during the light phase (9 AM-9 PM) for 12 wk. Liver lipid content and gene expression profile in the liver (Affymetrix 430 GeneChip with real-time PCR validation) were determined on completion of the exposure. CIH caused a 30% increase in triglyceride and phospholipid liver content (P < 0.05), whereas liver cholesterol content was unchanged. Gene expression analysis showed that CIH upregulated multiple genes controlling 1) cholesterol and fatty acid biosynthesis [malic enzyme and acetyl coenzyme A (CoA) synthetase], 2) predominantly fatty acid biosynthesis (acetyl-CoA carboxylase and stearoyl-CoA desaturases 1 and 2), and 3) triglyceride and phospholipid biosynthesis (mitochondrial glycerol-3-phosphate acyltransferase). A majority of overexpressed genes were transcriptionally regulated by sterol regulatory element-binding protein (SREBP) 1, a master regulator of lipogenesis. A 2.8-fold increase in SREBP-1 gene expression in CIH was confirmed by real-time PCR (P = 0.001). Expression of major genes of cholesterol biosynthesis, SREBP-2 and 3-hydroxy-3-methylglutaryl-CoA reductase, was unchanged. In conclusion, we have shown that CIH may exacerbate preexisting fatty liver of obesity via upregulation of the pathways of lipid biosynthesis in the liver.

Animals↗
PubMed1976

Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

NAD-specific "malic" enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO(4) (2-), suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn(2+), which cannot be replaced by NADP or Mg(2+). For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn(2+), and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 mumole/hr.mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO(2) for recycling during cessation of gas exchange in periods of severe drought.

Journal Article↗
PubMed1999

Production of multifunctional organic acids from renewable resources.

Recently, the microbial production of multifunctional organic acid has received interest due to their increased use in the food industry and their potential as raw materials for the manufacture of biodegradable polymers. Certain species of microorganisms produce significant quantities of organic acids in high yields under specific cultivation conditions from biomass-derived carbohydrates. The accumulation of some acids, such as fumaric, malic and succinic acid, are believed to involve CO2-fixation which gives high yields of products. The application of special fermentation techniques and the methods for downstream processing of products are described. Techniques such as simultaneous fermentation and product recovery and downstream processing are likely to occupy an important role in the reduction of production costs. Finally, some aspects of process design and current industrial production processes are discussed.

Aspartic Acid↗
PubMed2005

Effect of the conservation procedure on the contents of phenolic compounds and organic acids in chanterelle (Cantharellus cibarius) mushroom.

To check the influence of the conservation procedure in the chemical composition of chanterelle mushroom, phenolic compounds and organic acids of samples preserved under four different conditions (drying, freezing, conservation in olive oil and in vinegar) were determined. Phenolics and organic acids were analyzed by HPLC-DAD and HPLC-UV, respectively. The results showed that chanterelle is characterized by the presence of six phenolic compounds (3-, 4-, and 5-O-caffeoylquinic acid, caffeic acid, p-coumaric acid, and rutin) and five organic acids (citric, ascorbic, malic, shikimic, and fumaric acids). Samples preserved in olive oil also exhibited hydroxytyrosol, tyrosol, luteolin, and apigenin, whereas conservation in vinegar led to the detection of hydroxytyrosol, tyrosol, and tartaric acid in the analyzed samples. The conservation procedures to which chanterelle samples were subjected seem to affect the qualitative and quantitative phenolics and organic acids profiles.

Acetic Acid↗
PubMed1999

Tissue-specific effect of clofibrate on rat lipogenic enzyme gene expression.

Fibrate derivatives are commonly used to treat hyperlipidaemia; however, the mechanism of the antilipidaemic action of these drugs is still unknown. The effect of clofibrate (fibrate derivative) administration for 14 days on lipogenesis and on malic enzyme (EC 1.1.1.40) and fatty acid synthase (EC 2.3.1.85) gene expression in brown and white adipose tissues and in the liver was examined in rats. The rate of brown adipose tissue lipogenesis in the clofibrate-treated animals was significantly lower than that of the control rats. The rate of liver and white adipose tissue lipogenesis was not affected significantly by clofibrate. In brown adipose tissue, the drug treatment resulted in a depression of fatty acid synthase and malic enzyme mRNA levels. The fatty acid synthase mRNA level did not change significantly in the liver, whereas the malic enzyme mRNA level increased approximately 6-fold in this organ after clofibrate treatment. The malic enzyme mRNA level in white adipose tissue increased about 2-fold, while the fatty acid synthase mRNA level was unchanged after clofibrate feeding. The results presented in this paper provide further evidence that the hypolipidaemia caused by treatment of rats with clofibrate cannot be related to the inhibition of fatty acid synthesis in the liver and white adipose tissue. These data also indicate that clofibrate exhibits tissue specificity.

Adipose Tissue↗
PubMed2005

Analysis of organic acids in wines by Fourier-transform infrared spectroscopy.

Fourier-transform infrared (FTIR) spectroscopy has been a major point of development in many wine laboratories in recent years. It enables almost instant analysis of several properties of wine, usually with very good precision and accurate results. Nevertheless, validation procedures should not be forgotten and should be fully performed. Recovery experiments were performed by spiking wine samples with different amounts of organic acids (tartaric, malic, lactic, acetic and citric--the most prominent in wines). After FTIR analysis of the total acidity and of each organic acid concentration, recoveries were calculated. For total acidity recovery results were, in general, good and very close to 100% (64-111%). On the other hand, for individual organic acid concentrations, the recovery results were lower than 100% (11-73%) for all spiking additions. These results could be explained by spectroscopic interferences between the organic acids. Because they have similar infrared spectra, it is not easy to distinguish between them and, therefore, to achieve accurate calibration. When total acidity, with a different infrared spectrum from the other abundant compounds in the wine, was taken as a single property the recovery results were acceptable.

Acids↗
PubMed2003

Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates.

A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with (13)C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO(2) via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that alpha-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.

Acetates↗
PubMed1969

Oxidation D-malic and beta-alkylmalic acids wild-type and mutant strains of Salmonella typhimurium and by Aerobacter aerogenes.

A mutant strain of Salmonella typhimurium (SL 1634 dml-51) capable of growth on d-malate as sole carbon source was shown to produce d-malic enzyme. This enzyme was absent in the parent wild-type strain which was unable to grow on d-malate. Growth of the mutant on d-malate also resulted in a greatly increased level of beta-isopropylmalic enzyme compared with its level in the wild-type strain grown on citrate or l-malate. The d-malic and beta-isopropylmalic enzymes, both of which catalyze a nicotinamide adenine dinucleotide- and Mg(++)-dependent oxidative decarboxylation of their respective substrates, were shown to be distinct enzymes by selective inhibition with erythro-dl-beta-hydroxyaspartate and by other methods. Cell extracts of the mutant strain also oxidized dl-beta-methyl-, dl-beta-ethyl-, dl-beta-propyl- and dl-betabeta-dimethylmalates, in order of decreasing activity. dl-beta-Methyl-malate was shown to be oxidized by both the d-malic and the beta-isopropylmalic enzymes, whereas the oxidation of the other beta-alkylmalates appeared to be effected exclusively by the beta-isopropylmalic enzyme. beta-Isopropylmalic enzyme activity was induced by d-malate but not by l-malate, showing that it behaved as a d-malictype enzyme. Growth of Aerobacter aerogenes on d-malate, which caused induction of d malic enzyme, resulted in only a small increase in the activity of beta-isopropylmalic enzyme.

Aspartic Acid↗
PubMed2000

Lysine 199 is the general acid in the NAD-malic enzyme reaction.

Site-directed mutagenesis was used to change K199 in the Ascaris suum NAD-malic enzyme to A and R and Y126 to F. The K199A mutant enzyme gives a 10(5)-fold decrease in V and a 10(6)-fold decrease in V/K(malate) compared to the WT enzyme. In addition, the ratio for partitioning of the oxalacetate intermediate toward pyruvate and malate changes from a value of 0.4 for the WT enzyme to 1.6 for K199A, and repeating the experiment with A-side NADD gives isotope effects of 3 and 1 for the WT and K199A mutant enzymes, respectively. The K199R mutant enzyme gives only a factor of 10 decrease in V, and the pK for the general acid in this mutant enzyme has increased from 9 for the WT enzyme to >10 for the K199R mutant enzyme. Tritium exchange from solvent into pyruvate is catalyzed by the WT enzyme, but not by the K199A mutant enzyme. The Y126F mutant enzyme gives a 10(3)-fold decrease in V. The oxalacetate partition ratio and isotope effect on oxalacetate reduction for the Y126F mutant enzyme are identical, within error, to those measured for the WT enzyme. Thus, Y126 is important to the overall reaction, but its role at present is unclear. Data are consistent with K199 functioning as the general acid that protonates C3 of enolpyruvate to generate the pyruvate product in the malic enzyme reaction.

Alanine↗
PubMed1973

Organic Acid Changes in the Epidermis of Vicia faba and Their Implication in Stomatal Movement.

Considerable evidence indicates that the increase in guard cell turgor resulting in stomatal opening is brought about by active K(+) uptake into guard cells. Only a small increase in inorganic anions appears to accompany the increase in K(+). A plausible explanation is that organic acids are produced within guard cells and act as counterions, whereas the H(+) produced are exchanged for K(+).This hypothesis was tested by using different levels of ambient CO(2) in light to control stomatal aperture and at the same time measure changes in organic acid production in the epidermis of Vicia faba. Epidermal strips were used, quickly frozen in liquid nitrogen, and later extracted.A positive correlation was found between stomatal resistance (r(s), indirect measure of stomatal aperture) and CO(2) level. With decreases in r(s), total titratable acidity increased. The organic acids, glyceric, malic, and citric, in the epidermis, as measured by gas chromatography of trimethylsilyl derivatives, increased. Changes in glucose or sucrose were not found. These analyses provided evidence that organic acid production in the epidermis is associated with stomatal opening.

Journal Article↗
PubMed2002

Study of the organic acids composition of quince (Cydonia oblonga Miller) fruit and jam.

The organic acids present in several samples of quince fruit (pulp and peel) and quince jam (homemade and industrially manufactured) were analyzed by HPLC. The sample preparation was simple, involving only extraction with methanol (40 degrees C) and filtration through a Sep-pack C18 cartridge. The chromatographic separation was achieved using an ion exclusion column, Nucleogel Ion 300 OA (300 x 7.7 mm), in conjunction with a column heating device at 30 degrees C. An isocratic elution with H(2)SO(4) 0.01 N as the mobile phase, with a flow rate of 0.1 mL/min, and UV detection at 214 nm were used. These analyses showed that all samples presented a similar profile composed of at least six identified organic acids: citric, ascorbic, malic, quinic, shikimic, and fumaric acids. Several samples also contained oxalic acid. This study suggests that the organic acids levels and ratios may be useful for the determination of percent fruit content of quince jams. The citric acid value can also be used in the differentiation of the type of manufacture of the commercial quince jams (homemade or industrially manufactured).

Ascorbic Acid↗
PubMed1986

Discovery of an arachidonic acid C-8 lipoxygenase in the gorgonian coral Pseudoplexaura porosa.

The gorgonian coral Pseudoplexaura porosa contains a lipoxygenase capable of converting exogenous arachidonic acid into (8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid. The (8R)- (or 8-L-) configuration in this product, opposite to that observed in previously reported 8-lipoxygenase products, was determined unambiguously by comparison of oxidative ozonolysis fragments with authentic malic acid-derived standards. Extracts from the coral contained no detectable prostaglandins (PGAs, PGBs, PGEs, or PGFs). Although arachidonic acid represents one of the most abundant of the common fatty acids found in the phospholipid and total lipid fractions of P. porosa, products ascribable to the arachidonic acid 8-lipoxygenase pathway ((8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid, the corresponding alcohol 8-hydroxyeicosatetraenoic acid, further transformation products) have not yet been identified in the coral extracts. The physiological significance of the 8-lipoxygenase in this species remains a matter for speculation.

Animals↗
PubMed2006

The role of glucagon in regulating chicken hepatic malic enzyme and histidase messenger ribonucleic acid expression in response to an increase in dietary protein intake.

Increased dietary protein intake rapidly (3 h) decreases hepatic malic enzyme and increases hepatic histidase mRNA expression in broiler chicks. A series of experiments was conducted to determine the role that glucagon or a specific mixture of dietary amino acids might have in regulating the rapid changes in mRNA expression of these enzymes, when dietary protein intake is increased. Three hours after the injection of glucagon (240 microg/kg of BW) into the brachial vein of broiler chicks, hepatic malic enzyme mRNA expression was significantly lower and hepatic histidase mRNA expression was significantly greater than the level detected in saline-injected chicks. In addition, broiler chicks fed a high (40 g/ 100 g of diet) protein diet had significantly higher plasma glucagon levels at 1 and 3 h after initial access to this diet than broiler chicks fed a basal (22 g/100 g of diet) protein diet. The plasma glucagon concentration, however, was not different between the chicks fed the 2 dietary protein levels at 2 h after the initial access to the 2 diets. When a mixture of indispensable or dispensable amino acids was added to the basal diet to equal the concentrations of the individual indispensable or dispensable amino acids in the high protein diet, hepatic mRNA expression of malic enzyme and histidase were intermediate to the expression found in chicks fed the basal and high protein diet. The results indicate that glucagon may mediate the changes in the mRNA expression of malic enzyme and histidase in response to dietary protein intake and that total amino acid intake rather than the ingestion of specific amino acids regulates the mRNA expression of malic enzyme and histidase in chicks.

Animals↗
PubMed1992

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.

Acetyl-CoA Carboxylase↗
PubMed1991

Diurnal variations of lipogenic enzyme mRNA quantities in rat liver.

The diurnal variations in mRNA quantities of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase) in rat livers were detected. When the rats began feeding actively after lights out at 1900 h, the mRNA quantities were high from 0500 h to 0900 h in the morning. The variation in fatty acid synthase mRNA quantities was the most dramatic. However, no measurable variation in any enzyme levels including fatty acid synthase was detected. It may be because the half-lives of the enzymes are too long to be effected by the mRNAs which were high for several hours.

Acetyl-CoA Carboxylase↗
PubMed1992

Regulation of hepatic lipogenic enzyme gene expression by diet quantity in rats fed a fat-free, high carbohydrate diet.

This investigation concerns the effects of the level of intake of a high carbohydrate diet on transcriptional rate, mRNA concentration and enzyme induction for lipogenic enzymes in rat liver. Six hours after refeeding fasted rats, the transcriptional rates in livers reached low maximum levels with small quantities of diet, but the mRNA concentrations continued to increase as diet intake increased. Greater diet intake primarily increased transcriptional rates and mRNA concentrations of lipogenic enzymes. After refeeding for 16 h, the mRNA concentrations were sigmoidly increased relative to the diet quantity and reached maximum levels of 20-, 110-, 22- and 16-fold above each fasted level for acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase, respectively. After 3 d of refeeding (in a steady state of lipogenic enzyme activities), however, the transcriptional rates, mRNA concentrations and activity inductions of all the enzymes were sigmoidly increased relative to diet quantity, but were not different among the enzymes. Consequently, fatty acid synthesis and triglyceride levels in the liver were not increased by feeding less than 70% of ad libitum intake but were greatly increased by feeding greater than 70% of ad libitum intake.

Acetyl-CoA Carboxylase↗