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PubMed1993

Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes.

Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. beta-actin and albumin mRNA, as well as albumin secretion, were also assessed. Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.

Acetyl-CoA Carboxylase↗
PubMed1998

Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein.

A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17, respectively, was found to be much more stable in urea and thermal denaturation, but was enzymatically less active than the wild-type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased by approximately 10 degrees C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58 degrees C also conformed to a three-state model with the rate constant for the intermediate state of R9E/M17K (k2 = 0.03 min(-1)) being much smaller than the WT value (k2 = 2.39 min(-1)). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the K(mMn) and K(mMal) by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [k(cat)/(K(mMm)K(mNADP)K(mMal))] for the R9 mutants at least four orders of magnitude smaller than the WT. M17K has similar properties to the WT, while R9E is more labile than the WT enzyme. The above results indicate that the extra stability gained by the double mutant possibly occurs through the introduction of an extra ion-pair between E9 and K17, which freezes the double mutant in the putative intermediate state. Examination of the N-terminal amino acid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue. Since the R9E mutant, which has an extra Glu9-Lys15 ion-pair, is less stable than the WT, we conclude that the contribution to malic enzyme stability is specific for the Glu9-Lys17 ion-pair.

Amino Acid Sequence↗
PubMed1977

Influence of dietary lipid on lipogenic enzyme activities in coho salmon, Oncorhynchus kisutch (Walbaum).

Coho salmon (Oncorhynchus kisutch), 8 to 18 months of age, were maintained in culture tanks and were fed three semipurified diets. The diets contained 40% of energy from protein and 11.5%, 23%, or 46% of energy from lipid. The body weight gain and food conversion factors were similar among groups of fish fed the diets in each of the three experiments. Wet weight of mesenteric adipose tissue increased with increased amount of lipid in the diet; however, epaxial muscle lipid content was not influenced by the lipid content of the diet. Several hepatic and adipose tissue lipogenic enzymes (fatty acid synthetase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-isocitrate dehydrogenase) were assayed. These lipogenic enzymes exhibited high activities in liver and relatively low concentration in adipose tissue of the fish. The activities of all the hepatic lipogenic enzymes assayed, except for NADP-isocitrate dehydrogenase, were depressed as the level of lipid in the diet was increased; however, the activities of these enzymes in mesenteric adipose tissue were not influenced by the diets fed. The results of this study indicate that dietary lipid depresses hepatic lipogenic enzyme activities and that the liver may be a more important site for fatty acid synthesis than is adipose tissue in coho salmon.

ATP Citrate (pro-S)-Lyase↗
PubMed1988

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides.

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.

Aspartic Acid↗
PubMed2006

Campest-5-en-3-one, an oxidized derivative of campesterol, activates PPARalpha, promotes energy consumption and reduces visceral fat deposition in rats.

Dietary campest-5-en-3-one (campestenone), an oxidized derivative of campesterol, significantly reduced visceral fat weight and the concentration of triacylglycerol in serum and liver of rats. Dietary campestenone dramatically increased the activities and the mRNA expressions of mitochondrial and peroxisomal enzymes involved in beta-oxidation in the liver. Campestenone activated human peroxisome proliferator-activated receptor (PPAR) alpha as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. In contrast, dietary campestenone reduced the activities and the mRNA expressions of enzymes involved in fatty acid synthesis, except for the malic enzyme. Dietary campestenone decreased the sterol regulatory element binding protein-1 (SREBP-1) mRNA level. Energy expenditure was significantly higher in the feeding of campestenone in rats. Dietary campestenone reduced hepatic cholesterol concentration and increased fecal excretion of neutral steroids originated from cholesterol. Lymphatic absorption of cholesterol was reduced by the coadministration of campestenone in rats cannulated in the thoracic duct. These observations suggest a possibility that campestenone has an ability to prevent coronary heart disease by improving obesity and abnormality of lipid metabolism.

Animals↗
PubMed1977

Effects of fasting and feeding various diets on hepatic lipogenic enzyme activities in coho salmon (Oncorhynchus kisutch (Walbaum)).

The influence of fasting and diet composition on the time sequence of changes in hepatic lipogenic enzyme activities in coho salmon was investigated. Young coho salmon fed a high-carbohydrate diet for 3 weeks were then fasted for 2, 6, or 23 days. Liver preparations were assayed for fatty acid synthetase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. Fasting the fish for 2 or 6 days did not influence the activities of these enzymes; however, by the end of the 23-day fast the activities of all these enzymes had decreased. Changing the diet of the fish from high-carbohydrate to high-fat had only a minimal influence on the activities of the hepatic lipogenic enzymes after 7 to 14 days. Longer-term studies demonstrated that high-fat diets did eventually depress lipogenic enzyme activities. The effect of fasting and feeding on the level of lipogenic enzyme activities was observed only after several weeks, in contrast to hours in the rat. This may reflect a difference between poikilothermous and homoiothermous animals.

ATP Citrate (pro-S)-Lyase↗
PubMedDate not supplied

Developmental changes of lipogenic enzyme activities and lipogenesis in brown adipose tissue and liver of the rat.

1. Brown adipose tissue (BAT) and liver lipogenesis in vivo estimated by using 3H2O as tracer was very low and did not change significantly between 10 and 20 days after birth. Lipogenesis increased dramatically in both tissues by weaning at 20 days, peaking between 25 and 30 days of age. Since that time the rate of fatty acid synthesis in BAT decreased gradually to reach adult level after 2 months, whereas in the liver there was a sharp decrease of lipogenesis. 2. The activities of fatty acid synthase, citrate cleavage enzyme, malic enzyme and glucose 6-phosphate dehydrogenase essentially followed a similar course of developmental changes as lipogenesis. 3. In contrast to the enzymes listed above NADP-linked isocitrate dehydrogenase remained unaltered over the period studied, whereas lactate and malate dehydrogenases exhibited very high activity at 10 days after birth and from then decreased to reach adult level at the age of about 20 days. 4. The data obtained indicate that no substantial differences could be detected in the developmental pattern of lipogenesis and lipogenic enzyme activities between BAT and liver up to 30 days of age but after this time these processes were not co-ordinated in both tissues. Beyond this time the BAT was characterized by a much higher rate of lipogenesis than the liver. 5. The results are discussed in terms of the nutrient changes and the relationship between thermogenesis and lipogenesis in BAT.

Adipose Tissue, Brown↗
PubMed2000

Carbon and nitrogen metabolism in Rhizobium.

One of the paradigms of symbiotic nitrogen fixation has been that bacteroids reduce N2 to ammonium and secrete it without assimilation into amino acids. This has recently been challenged by work with soybeans showing that only alanine is excreted in 15N2 labelling experiments. Work with peas shows that the bacteroid nitrogen secretion products during in vitro experiments depend on the experimental conditions. There is a mixed secretion of both ammonium and alanine depending critically on the concentration of bacteroids and ammonium concentration. The pathway of alanine synthesis has been shown to be via alanine dehydrogenase, and mutation of this enzyme indicates that in planta there is likely to be mixed secretion of ammonium and alanine. Alanine synthesis directly links carbon catabolism and nitrogen assimilation in the bacteroid. There is now overwhelming evidence that the principal carbon sources of bacteroids are the C4-dicarboxylic acids. This is based on labelling and bacteroid respiration data, and mutation of both the dicarboxylic acid transport system (dct) and malic enzyme. L-malate is at a key bifurcation point in bacteroid metabolism, being oxidized to oxaloacetate and oxidatively decarboxylated to pyruvate. Pyruvate can be aminated to alanine or converted to acetyl-CoA where it either enters the TCA cycle by condensation with oxaloacetate or forms polyhydroxybutyrate (PHB). Thus regulation of carbon and nitrogen metabolism are strongly connected. Efficient catabolism of C4-dicarboxylates requires the balanced input and removal of intermediates from the TCA cycle. The TCA cycle in bacteroids may be limited by the redox state of NADH/NAD+ at the 2-ketoglutarate dehydrogenase complex, and a number of pathways may be involved in bypassing this block. These pathways include PHB synthesis, glutamate synthesis, glycogen synthesis, GABA shunt and glutamine cycling. Their operation may be critical in maintaining the optimum redox poise and carbon balance of the TCA cycle. They can also be considered to be overflow pathways since they act to remove or add electrons and carbon into the TCA cycle. Optimum operation of the TCA cycle has a major impact on nitrogen fixation.

Alanine↗
PubMed1998

Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme.

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring beta-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

Ethanol↗
PubMed2000

Development and validation of a capillary electrophoresis method for the measurement of short-chain organic acids in natural rubber latex.

Short-chain organic acid contents in serum of natural latex are interesting to measure and capillary electrophoresis (CE) has proved to be a good tool for their study. In the present work a method has been developed to identify the short-chain organic acids present in sera of natural rubber latex (oxalic, formic, fumaric, aconitic, succinic, malic, glutaric, citric, acetic, glycollic, propionic and quinic acids), the separation was optimised and the quantification method validated. The separation was performed on a CE system with UV detection at 200 nm. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm I.D.) and was operated at -10 kV potential. The separation buffers were prepared with 0.5 M H3PO4, 0.5 mM cetyltrimethylammonium bromide and pH adjusted by adding NaOH to 6.25 except for propionic acid which was better measured at pH 7.00. Validation parameters are adequate and limits of detection range from 0.005 mM to 1.6 mM. Short-chain organic acids were measured with this method in sera of three different types of latex.

Acids↗
PubMed2003

Isozymes in Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids and Ae. kotschyi x S. cereale amphiploids in relation to their parents.

Seven enzymatic systems in F1 Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids, Aegilops kotschyi x S. cereale amphiploids and their parental species (Ae. kotschyi, Ae. biuncialis and S. cereale) were analysed by starch and polyacrylamide gel electrophoresis. Five of them (phosphoglucose isomerase, glutamic oxalacetic transaminase, esterase, acid phosphatase, and diaphorase) were polymorphic and two (malic dehydrogenase and superoxide dismutase) were monomorphic. Several isophorms of phosphoclucose isomerase, esterase, acid phosphatase, and diaphorase were detected in some hybrids and amphiploids, but absent in the parents. The role of regulators, translocations and recombination is discussed in relation to the origin of these new isophorms. Some parental isozymes were absent both in hybrids and amphiploids, probably as a result of the suppression of structural genes in new combinations of the three genomes.

Acid Phosphatase↗
PubMed2000

An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries.

The compartmentation of key processes in sugar, organic acid and amino acid metabolism was studied during the development of the flesh and seeds of grape (Vitis vinifera L.) berries. Antibodies specific for enzymes involved in sugar (cell wall and vacuolar invertases, pyrophosphate: fructose 6-phosphate phosphotransferase, aldolase, NADP-glyceraldehyde-P dehydrogenase, cytosolic fructose 1,6-bisphosphatase), photosynthesis (Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase), amino acid metabolism (cytosolic and mitochondrial aspartate aminotransferases, alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase), organic acid metabolism (phosphoenolpyruvate carboxylase, NAD- and NADP-dependent malic enzyme, ascorbate peroxidase), and lipid metabolism (acetyl CoA carboxylase, isocitrate lyase) were used to determine how their abundance changed during development. There were marked changes in the abundance of many of these enzymes in both the flesh and seeds. The intercellular location of some enzymes was investigated using immunohistochemistry. Several enzymes (e.g. phosphoenolpyruvate carboxylase and those involved in amino acid metabolism) were associated with tissues likely to function in the transport of imported assimilates, such as the vasculature. Although other enzymes (e.g. NADP-malic enzyme and soluble acid invertase, involved in the metabolism of sugars and organic acids) were largely present in the parenchyma cells of the flesh, their distribution was extremely heterogeneous. This study shows that when considering the metabolism of complex structures such as fruit, it is essential to consider how metabolism is compartmentalized between and within different tissues, even when they are apparently structurally homogeneous.

Amino Acids↗
PubMed2003

Validated capillary electrophoresis method for small-anions measurement in wines.

A capillary electrophoresis method has been developed and validated for acetic, citric, fumaric, lactic, malic, oxalic, succinic, and tartaric acids plus the measurement of nitrate and sulfite ions in white and red wines. The separation was carried out in a neutral coated capillary. Separation was performed at -14 kV of applied potential. Temperature was maintained at 20 degrees C. The background electrolyte used was 200 mM phosphate buffer at pH 7.50. Separation was obtained in less than 13 min. Validation parameters obtained for the method permit it to be considered adequate for routine analysis.

Anions↗
PubMed1979

Enzyme histochemical characteristics of human and kitten odontoclasts and kitten osteoclasts: a comparative study using whole cells.

Methods for the histochemical demonstration of enzymes in whole cell preparations of odontoclasts and osteoclasts are described. Enzyme histochemical characteristics of human and kitten odontoclasts from resorbing primary teeth and of osteoclasts from kitten femur metaphyses were determined and compared. The enzyme profiles, times for the appearance of detectable reaction product, intensity of the reactions and localization of the reaction products were similar in all three types of giant cell. These findings suggest that odontoclasts have enzyme properties and metabolic functions similar to those of osteoclasts. Species differences appear to be minor, although the NADP-dependent enzymes are less active in human than in kitten odontoclasts. Both odontoclasts and osteoclasts are rich in enzymes concerned with energy production and possess considerable activity of enzymes usually associated with catabolic functions. Metabolic pathways are well developed in respect of the utilization of succinic, malic, glutamic, lactic and isocitric acids, beta-hydroxybutyric acid and glucose-6-phosphate, and they also possess phosphatases, non-specific esterases and leucine naphthylamidase. The distribution of enzyme reaction products for the individual enzymes demonstrated is consistent with the presence in these cells of large numbers of mitochondria and lysosome-like organelles. Considerable phosphatase activity is demonstrable in both odontoclasts and osteoclasts at both neutral and acid pH.

Animals↗
PubMed1998

Ozonation Products of Organosolvolytic Extracts from Vegetal Materials.

The ozonation of lignin in an organic solution was used to produce oxyaromatic compounds. Corn (Zea mays) stalk and poplar (Populus deltoides) wood lignin solutions (organosolvolytic juices) obtained from their respective autohydrolyzed materials and an organosolv lignin were treated with ozone, and the time course trend of the identified reaction products was studied. GC/MS and GC/FID were used to identify and quantify (internal standard method) the reaction products. The following acids were identified: glycolic, oxalic, malic, glyceric, p-hydroxybenzoic, and vanillic. In addition, p-hydroxybenzaldehyde, vanillin, syringaldehyde, and hydroquinone were also identified. All the oxyaromatic aldehydes reached maximum yields at short reaction times (between 1 and 8 min). These results were used to determine the ratio between the different phenylpropane units of their constitutive lignins. The ratio of p-hydroxyphenyl:guaiacyl:syringyl units for corn stalk, poplar wood, and organosolv lignins was 5.3:2:1, 0.1:1:1.1, and 0:1:1.1, respectively.

Journal Article↗
PubMed2002

Studies aimed at the total synthesis of the antitumor antibiotic cochleamycin A. An enantioselective biosynthesis-based pathway to the AB bicyclic core.

[reaction: see text] A convergent, highly enantioselective synthesis of the fully functionalized AB sector of cochleamycin A is described. A pair of building blocks, crafted from L-malic and L-ascorbic acids, are conjoined in a manner that gives rise to an (E,Z,E)-1,6,8-nonatriene. On heating, the latter undergoes stereocontrolled intramolecular Diels-Alder cyclization via an endo transition state.

Antibiotics, Antineoplastic↗
PubMed2006

Effect of exercise on hepatic gene expression in an obese mouse model using cDNA microarrays.

To understand the molecular mechanisms involved in the effect of exercise training, we examined hepatic transcriptional profiles using cDNA microarrays in exercise-trained and untrained mice with diet-induced obesity. C57BL/6J male mice (n = 10/group) were fed with a normal diet, high-fat diet (HFD), or HFD with exercise training for 12 weeks. The expression level of approximately 10,000 transcripts in liver tissues from each group was assessed using cDNA microarray analysis. Exercise training improved lipid profiles and hepatic steatosis and decreased body fat mass induced by the HFD. Seventy-three genes were differentially expressed in the HFD- and/or HFD with exercise training-treated groups, compared with the normal diet- and HFD-fed groups, respectively. Interestingly, the expression profiles involved in metabolism, such as elongation of very long chain fatty acids-like 2, lipin, and malic enzyme, were changed by exercise training. In addition, expression of genes altered by exercise training related to defense and stress response, including metallothionein 1 and 2 and heat shock protein, showed interesting findings. Our study showed beneficial effects of exercise training in preventing the development of obesity and metabolic disorders in mice with diet-induced obesity.

Analysis of Variance↗