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PubMed1994

Cloning and expression of pigeon liver cytosolic NADP(+)-dependent malic enzyme cDNA and some of its abortive mutants.

A full-length 1927-base-pair cDNA of pigeon liver malic enzyme was obtained by utilizing the screening of the cDNA library and polymerase chain reaction techniques. The cDNA contained one open reading frame coding for 557 amino acid residues, flanked by 86 and 167 nucleotides at the 5' and 3' termini, respectively, and was successfully cloned and expressed in Escherichia coli cells. Functionally active recombinant malic enzyme was purified from the cells. This recombinant enzyme has a Km value for L-malate of 160 +/- 30 microM, which is almost identical to that for the natural enzyme (150 +/- 17 microM). The Km value for Mn2+ (4.2 +/- 0.3 microM) is higher than that for the natural pigeon malic enzyme (1.4 +/- 0.2 microM), while the Km value for NADP+ (3.8 +/- 0.3 microM) is lower than that for the natural enzyme (10.8 +/- 0.1 microM). The catalytic constant (kcat) for the recombinant enzyme is decreased by 3.6-fold, but the substrate inhibition constant for L-malate is increased by about 40-fold. Change in the quaternary structure of the recombinant enzyme was revealed in the pH perturbation examination. A truncated pigeon liver malic enzyme, lacking the first 13 amino acid residues, and a recombinant protein, mutated at F19S, N250S, and L353Q, showed no enzymatic activity. Both abortive recombinant mutant proteins were still able to bind with 2',5'-ADP agarose; however, the fluorescence emission spectrum of the protein bound NADPH did not show a blue shift as the natural enzyme. In accordance with these observations, we suggest that the adenosine 2',5'-bisphosphate binding domain of the NADP+ binding site in the beta alpha beta motif may still be retained in these mutant proteins. However, the local hydrophobic environment for the binding of the nicotinamide moiety of the coenzyme molecule may be altered. Therefore, the lack of catalytic activity of the mutant proteins could be attributed to an improper orientation of the bound NADP+.

Adenosine Diphosphate↗
PubMedDate not supplied

Effect of catecholamines on enzyme release from human adipose tissue cells.

Effect of adrenaline (A) and noradrenaline (NA) on the release of enzymes were studied in the isolated human adipocytes incubated in Krebs-Hanseleit buffer to which albumins saturated with fatty acids or albumins, from which fatty acids were partly removed, were added. In the incubation fluid the activity of lactic dehydrogenase, malic dehydrogenase, isocitrate dehydrogenase or pyruvate kinase was determined. Besides that, the effects of adrenaline and noradrenaline on the content of non-esterified fatty acids (NEFA) in the adipocytes and on the release of glycerol into the incubation fluid were studied. Both A and NA stimulated the release of all examined enzymes into the incubation medium. The amount of the released enzymes, determined on the basis of changes in their activity in the incubation medium, depended on the degree of albumin saturation with fatty acids. Increased release of enzymes into the incubation fluid was associated with a rise in the content of NEFA in the adipocytes. Gycerol release into the incubation medium was independent of the degree of albumin saturation with fatty acids.

Adipose Tissue↗
PubMed1967

Experimental sprue-like small intestinal lesions in pigs.

The virus of transmissible gastroenteritis produced sprue-like lesions in the small intestines of young pigs. These lesions were characterized by villous shortening, fusing and blunting in the jejunum and ileum. There was decreased height of the brush border and morphologic alteration of the villous epithelial cells from simple columnar to a variable cuboidal type. Accompanying these microscopic lesions were histochemical changes characterized by decreased staining intensity of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, leucine aminopeptidase, succinic dehydrogenase and malic dehydrogenase in the affected intestinal mucosa. The clinical nature of transmissible gastroenteritis in the pig together with the histopathologic and histochemical changes may provide a useful experimental model for obtaining additional basic information on enteric disturbances.

Acid Phosphatase↗
PubMed1980

Identification of shellfish fatty acids and their effects on lipogenic enzymes.

The fatty acids which are common to and characteristic of shellfish, were identified by mass spectrometry and NMR spectral analyses as being: octadecatetraenoioc acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid. When the fatty acids isolated by high performance liquid chromatography were separately intubated into rats, hepatic glucose-6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40) and acetyl-CoA carboxylase (EC 6.4.1.2) were reduced more effectively as compared with linoleic acid intubation. These enzymes were reduced most markedly by eicosapentaenoic acid-intubation. The fatty acids seem to be effective components in reduction of triacylglycerol and lipogenic enzyme levels in rats fed on shellfish.

Acetyl-CoA Carboxylase↗
PubMed1993

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme.

The nucleotide sequence of a full-length cDNA encoding NAD(+)-malic enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2269 bases comprises a 5'-leader, a single open reading frame of 1851 bases, and the complete 3'-noncoding region of 340 bases. The first 12 amino acids of the translated sequence are hydrophobic, typical of mitochondrial translocation signals, and do not appear in the purified mature protein. The mature protein contains 605 amino acids and has a molecular mass of 68,478 Da. The amino acid sequences of tryptic peptides from the purified protein and also the N-terminal sequence show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the ascarid protein with the human and rat liver NAD(+)-malic enzymes reveals highly conserved regions interrupted with long stretches of lesser homologous sequences. Structural motifs such as the putative nucleotide binding domains and also the malate binding site are clearly identified by alignment of the three protein sequences.

Amino Acid Sequence↗
PubMed1963

STUDIES ON LYSOSOMES. III. THE EFFECTS OF STREPTOLYSINS O AND S ON THE RELEASE OF ACID HYDROLASES FROM A GRANULAR FRACTION OF RABBIT LIVER.

Streptolysins O and S from hemolytic streptococci have been added to granular fractions of tissue homogenates in 0.25 M sucrose prepared from rabbit liver, heart, spleen and lymph nodes. At concentrations ranging from 0.65 microg/ml to 2.0 mg/ml of streptolysin S, and from 10 microg/ml to 1.0 mg/ml of streptolysin O, two lysosomal enzymes (beta glucuronidase and acid phosphatase) and, to a lesser degree, one mitochondrial enzyme (malic dehydrogenase) were released into the supernatants of the reaction mixture. Although the hemolytic action of each lysin paralleled the effect on lysosomes, at equivalent levels of hemolytic activity, SLS was approximately 10 times more active on lysosomes than SLO. SLO was inhibited by cholesterol, cortisol, and specific antibody in vitro; pretreatment of animals with cortisone decreased the susceptibility of their isolated lysosomes to SLO. These agents failed to prevent SLS action on lysosomes. SLO had a pH optimum of 6.5 against lysosomes while SLS was maximally active at 7.5. No other streptococcal extracellular products were as active on lysosomes as the streptolysins, although activated streptococcal proteinase precursor released some hydrolases from the granules. Similarities between the actions of SLO and SLS on red cells and lysosomes suggested that the membranes bounding lysosomes and erythrocytes have common properties.

Acid Phosphatase↗
PubMed2006

Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production.

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.

Anaerobiosis↗
PubMed1985

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism. Review.

Branched-chain amino acid metabolism in skeletal muscle promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.

Alanine↗
PubMed2005

Dietary protein source affects lipid metabolism in the European seabass (Dicentrarchus labrax).

The study was undertaken to evaluate the effects of dietary protein sources on lipogenesis and fat deposition in a marine teleost, the European seabass (Dicentrarchus labrax). Four isonitrogenous (crude protein (CP, Nx6.25), 44% DM) and isoenergetic (22-23 kJ/g DM) diets were formulated to contain one of the following as the major protein source: fish meal (FM), one of two soy protein concentrates (SPC) and corn gluten meal (CGM). Apparent digestibility coefficients of the diets and raw ingredients, as well as soluble nitrogen (ammonia and urea) and phosphorus excretion were measured. Growth rates of seabass fed plant protein-based diets were significantly lower than those fed fish meal based diet. The protein utilisation was strongly correlated to the dietary essential amino acids index. Measurements of N excretion (ammonia and urea nitrogen) confirmed these data. Daily fat gain at the whole body level ranged between 1.1 to 1.7 g/kg BW, with the highest values being recorded in fish fed the fish meal based diet. Levels of plasma triglycerides and cholesterol were lower in fish fed soy protein diets than in those fed the diet solely based on fish meal. Soy protein rich diets decreased the activities of selected hepatic lipogenic enzymes (glucose 6-phosphate dehydrogenase, malic enzyme, ATP-citrate lysase, acetylcoenzyme A carboxylase and fatty acid synthetase). Highest lipogenic enzyme activities where found in fish fed the fish meal diet, except for fatty acid synthetase which was increased in seabass fed the corn-gluten meal based diets. Overall data suggest that dietary protein sources affects fat deposition and the lipogenic potential in European seabass.

Animals↗
PubMed1982

Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone.

The lipogenic capacity of rat liver is increased in animals fed a high carbohydrate, fat-free diet or by the administration of 2,2',5'-triiodo-L-thyronine. Underlying this change is a generalized induction of the enzymes involved in lipogenesis, including glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, which together serve to generate the additional NADPH required for increased fatty acid synthesis. This report presents evidence indicating that induction of the hexose-shunt dehydrogenases involves increased enzyme synthesis secondary to elevated enzyme specific mRNA levels, as has previously been shown for malic enzyme. Activities of specific mRNAs, estimated by cell-free translation of hepatic poly(A)-containing RNA in the mRNA dependent rabbit reticulocyte lysate, were compared with enzyme specific activities and relative rates of specific enzyme synthesis. The 2-fold increase in glucose-6-phosphate dehydrogenase specific activity in hyperthyroid rats and the 13-fold increase in rats fed a high carbohydrate, fat-free diet, relative to euthyroid, chow-fed controls were paralleled by comparable increases in the synthetic rates and mRNA levels of this enzyme. Similarly, consonant changes in the rate of enzyme synthesis and concentration of 6-phosphogluconate dehydrogenase mRNA accompanied the 2.5- and 3-fold increases in specific activity of this enzyme observed in response to hormonal and dietary induction, respectively. Thus, both thyroid hormone and carbohydrate feeding appear to induce glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase primarily by increasing the effective cellular concentrations of their respective mRNAs and, consequently, their rates of synthesis.

Animals↗
PubMed1995

Dietary omega 6 fatty acids and the effects of hyperthyroidism in mice.

The influence of the type of dietary fat on the effects of thyroid hormones was investigated in mice. Hyperthyroidism was achieved by providing thyroid hormones (T3 and T4) in the drinking water. Both hyperthyroid and euthyroid mice (Mus musculus) were fed isoenergetic diets containing 18% (w/w) total lipid but differing in fatty acid composition. Diets were either low in the polyunsaturated linoleic acid (18:2, omega 6) and high in saturated fatty acids (SFAs) or low in saturated fats and high in the polyunsaturated fatty acid (PUFA), linoleic acid. Treatments were maintained for 21-22 days. Plasma thyroid hormone levels, standard metabolic rate (SMR), changes in body mass, specific activities of malic enzyme (ME), Na-K-ATPase and glycerolphosphate dehydrogenase (GPDH) of the liver were measured. Fatty acid composition of the liver phospholipids was also determined. Levels of T3 (15-17 nM) and T4 (250-255 nM) were significantly higher in the respective hyperthyroid groups. There was no significant influence of the diet on hormone levels. Hyperthyroidism increased the SMR 37-44% above the euthyroid levels. A significant body weight loss of 14-18% was observed in hyperthyroid mice on the PUFA diet but not in those on the SFA diet. PUFA diet significantly reduced the activity of ME but had no effect on Na-K-ATPase or GPDH activity. Activities of Na-K-ATPase and GPDH were significantly elevated in all hyperthyroid groups. Mice on T4 and PUFA diet showed a highly significant 399% increase in GPDH activity above the euthyroid level.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

The influence of metal ions on malic enzyme activity and lipid synthesis in Aspergillus niger.

In the presence of copper significant induction of citric acid overflow was observed, while concomitantly lower levels of total lipids were detected in the cells. Its effect was more obvious in a medium with magnesium as sole divalent metal ions, while in a medium with magnesium and manganese the addition of copper had a less pronounced effect. Since the malic enzyme was recognised as a supplier of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate for lipid biosynthesis, its kinetic parameters with regard to different concentrations of metal ions were investigated. Some inhibition was found with Fe(2+) and Zn(2+), while Cu(2+) ions in a concentration of 0.1 mM completely abolished malic enzyme activity. The same metal ions proportionally reduced the levels of total lipids in Aspergillus niger cells. A strong competitive inhibition of the enzyme by Cu(2+) was observed. It seemed that copper competes with Mg(2+) and Mn(2+) for the same binding site on the protein.

Aspergillus niger↗
PubMed2006

Differential regulation of intestinal lipid metabolism-related genes in obesity-resistant A/J vs. obesity-prone C57BL/6J mice.

The effects of high-fat (HF) feeding on gene expression in the small intestine were examined using obesity-resistant A/J mice and obesity-prone C57BL/6J (B6) mice. Both strains of mice were maintained on low-fat (LF; 5% fat) or HF (30% fat) diets for 2 wk. Quantitative reverse transcription-PCR analysis revealed that lipid metabolism-related genes, including carnitine palmitoyltransferase (CPT) I, liver fatty acid binding protein, pyruvate dehydrogenase kinase-4, and NADP(+)-dependent cytosolic malic enzyme, were upregulated by HF feeding in both strains of mice. The upregulated gene expression levels were higher in A/J mice than in B6 mice, suggesting more active lipid metabolism in the small intestine of A/J mice. The prominent upregulation of the lipid metabolism-related genes were specific to the small intestine; the expression levels were little or unchanged in the liver, muscle, and white adipose tissue. The increase by HF feeding and predominant expression of the intestinal lipid metabolism-related genes in A/J mice were reflected in the enzyme activities; malic enzyme, CPT, and beta-oxidation activities were increased by HF feeding, and the upregulated malic enzyme and CPT activities were significantly higher in obesity-resistant A/J mice compared with those in obesity-prone B6 mice. These findings suggest that intestinal lipid metabolism is associated with susceptibility to obesity.

Adipose Tissue↗
PubMed1987

The NADPH consumption regulates the NADPH-producing pathways (pentose phosphate cycle and malic enzyme) in rat adipocytes.

The changes in the activity of the pentose phosphate cycle and the malic enzyme produced by the activation or inhibition of different NADPH-consuming pathways have been studied. The inhibition of the fatty acid synthesis by kynurenate produced a decrease in the flux through the pentose phosphate cycle and a diminution in the malic enzyme pathway. The incubation of the adipocytes in the presence of ter-butyl-hydroperoxide, a compound which is metabolized via a NADPH-consuming pathway, produced a big increase in the pentose phosphate cycle and the malic enzyme activities. The regulation of these NADPH-producing pathways by the NADPH/NADP ratio is discussed.

Adipose Tissue↗
PubMed1998

Ergosteroids III. Syntheses and biological activity of seco-steroids related to dehydroepiandrosterone.

The unusual activity of some D-ring-seco estrogens led us to prepare several seco steroids related to dehydroepiandrosterone (DHEA) and to test for their ability to mimic thyroid hormone and 7-oxo-DHEA (1) as inducers of thermogenic enzymes in rats' livers. Only one, 3 beta-acetoxy-17a-oxa-androst-5-ene-7,17-dione (17), was capable of inducing both mitochondrial glycerophosphate dehydrogenase and malic enzyme. The closely related 3 beta-hydroxy-17a-oxa-androsta-5,15-diene-7,17-diones (both 14 alpha and 14 beta, 14 and 15) induce the formation of malic enzyme but not of glycerophosphate dehydrogenase. The 3 beta-propionyl ester of the above 14 alpha steroid was not active, presumably because it was not deacylated in vivo. The 16,17 dicarboxylic acid (9) produced by opening the D-ring also induced the formation of malic enzyme but not of glycerophosphate dehydrogenase. 3 beta-Acetoxyandrost-5-ene-7,16,17-trione, an intermediate in the synthesis of D-ring seco compounds enhanced the formation of both enzymes. Twelve other D-ring seco compounds were not active. Seco androstanes oxygenated at position 7 and with expanded A or B rings were not active.

Animals↗
PubMed1988

Differentiation in culture of cells from an avian holocrine secretory gland: preparation of isolated cells and conditions which induce accumulation of malic enzyme.

Mammalian sebaceous glands contain cells which are constantly going through a process of cell division, differentiation, and destruction. Birds have an analogous holocrine secretory gland, the uropygial gland, which is an excellent model for mammalian sebaceous glands and for analysis of the regulation of differentiation. Isolated uropygial cells were purified in good yield, and with high viability, after enzymatic digestion of the duck uropygial gland. Almost exclusively progenitor (basal) cells are recovered after separation of isolated cells on a Percoll density gradient; mature uropygial cells are destroyed during preparation of isolated cells. In primary culture, uropygial gland cells grow to confluence and partially duplicate the in vivo differentiation pathway. Malic enzyme activity increases 30-fold during 4 wks in culture, but there is little, if any, accumulation of fatty acid synthase and only a modest deposition of fat droplets. Medium conditioned by chick embryo fibroblasts inhibits the accumulation of malic enzyme without affecting cell growth. The basement membrane components, collagen, laminin, and Matrigel, which stimulate differentiation in other cell systems, were without effect on uropygial gland cultures. Triiodothyronine, cyclic AMP, and dexamethasone together with isobutylmethylxanthine had no effect on cell growth or malic enzyme activity. Epidermal growth factor, which stimulates cell division, increased cell number with no increase in malic enzyme accumulation. Factors which would stimulate further differentiation are missing from our culture system, but may include components of the basal lamina and/or factors secreted by mesenchymal cells.

1-Methyl-3-isobutylxanthine↗