Showing papers on "Malic enzyme published in 1994"
TL;DR: A new regulatory element for peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers was found in the promoter of the malic enzyme gene, suggesting a broader function for the PPAR in the regulation of lipid metabolism.
156 citations
Journal Article•
TL;DR: The complete nucleotide sequence of a 2300-base pair alpha subunit cDNA clone showed that this gene encodes a protein that contains all of the motifs required for a complete and functional malic enzyme.
Abstract: In some C4 plant species, a mitochondrial NAD-dependent malic enzyme (EC 1.1.1.39) (NAD-ME) catalyzes the decarboxylation of 4 carbon malate in the bundle sheath cells, releasing CO2 for the Calvin cycle of photosynthesis. In amaranth, a dicotyledonous NAD-ME-type C4 plant, the photosynthetic NAD-ME purified as two subunits of 65 and 60 kDa, designated alpha and beta, respectively. Antiserum raised against the alpha subunit reacted only with the 65-kDa protein in immunoblot analysis. Immunogold electron microscopy using the alpha subunit antiserum demonstrated that this protein was localized specifically to the mitochondrial matrix of bundle sheath cells. The complete nucleotide sequence of a 2300-base pair alpha subunit cDNA clone showed that this gene encodes a protein that contains all of the motifs required for a complete and functional malic enzyme. The alpha subunit has significant similarity along its entire length to other known NAD- and NADP-dependent malic enzymes from plants, animals, and bacteria. The findings presented here provide new insights about the C4 photosynthetic NAD-ME and its evolutionary relationship to other forms of malic enzyme present in eukaryotic and prokaryotic organisms.
72 citations
TL;DR: In some C4 plant species, a mitochondrial NAD-dependent malic enzyme (EC 1.1.39) catalyzes the decarboxylation of 4 carbon malate in the bundle sheath cells, releasing CO2 for the Calvin cycle of photosynthesis as discussed by the authors.
71 citations
TL;DR: NADP+‐ME found in tumor cells does not constitute a tumor‐specific isoform as suggested by other studies but is identical to the ‘housekeeping protein’ predominantly expressed in human liver and white adipose tissue.
61 citations
TL;DR: Northern blot analysis indicates that the expression of this gene is regulated by external stress such as submergence, and the deduced amino acid sequence coded for by the cDNA indicates a high level of homology to chloroplast type NADP-ME.
Abstract: We have isolated a rice cDNA clone that is homologous to the gene for the maize NADP-dependent malic enzyme (EC 1.1.1.40; NADP-ME). The deduced amino acid sequence coded for by the cDNA indicates a high level of homology to chloroplast type NADP-ME, including a transit peptide with pronounced hydrophobic properties at the amino terminus. Northern blot analysis indicates that the expression of this gene is regulated by external stress such as submergence.
47 citations
TL;DR: Sequence analysis of a 4·6‐kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe revealed the presence of an open reading frame, coding for a 565 amino acid polypeptide.
Abstract: Sequence analysis of a 4.6-kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide. The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2.0 kb. The mae2 gene was mapped on chromosome III by chromoblotting. The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins. Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes. Two of these regions have previously been reported to be NAD- and NADP-binding sites.
47 citations
TL;DR: Electroanalytical methods based on biosensor technology and differential pH measurement for the determination of l-lactic and l-malic acids in wine were developed and results correlated well when compared with those obtained with the commonly used spectrophotometric procedure.
43 citations
TL;DR: The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms.
Abstract: Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.
43 citations
TL;DR: Northern blot analysis showed a strong tissue-specific transcription with a predominantly high expression-rate in organs with a low division-rate and comparison of the amino acid sequence of this isoform with published sequences of other human ME isoforms shows stretches of homology interrupted by larger regions with significant differences.
Abstract: Mitochondrial NADP(+)-dependent malic enzyme (ME; EC 1.1.1.39) has been purified to homogeneity and characterized kinetically from bovine heart. Partial amino acid sequence information allowed amplification of a specific bovine cDNA, which was used to isolate a full-length human cDNA of this isoform of ME. The cDNA is 1930 bp long and codes for a protein of 604 amino acids. Comparison of the amino acid sequence of this isoform with published sequences of other human ME isoforms shows stretches of homology interrupted by larger regions with significant differences. The human protein has been expressed in Escherichia coli, and the recombinant human protein has the same kinetic properties as the corresponding protein purified from bovine heart. Northern blot analysis showed a strong tissue-specific transcription with a predominantly high expression-rate in organs with a low division-rate.
41 citations
TL;DR: The results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation.
Abstract: Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.
37 citations
TL;DR: The 59- and 62-kDa subunits of the mitochondrial NAD+-dependent malic enzyme were purified from Solanum tuberosum L. (potato) and revealed a simple pattern of hybridization for both subunits, indicating a simple gene structure or small number of genes encoding the two subunits.
TL;DR: In this paper, the effects of a decrease in dietary protein level on the kinetic behavior of the four NADPH production systems [glucose 6-phosphate dehydrogenase (G6PDH), 6phosphogluconate de-decrease (6PGDH), malic enzyme (ME), and NADP-isocitrate dehydrogenases (NADP-IDH)] were investigated in four different trout tissues.
TL;DR: It is suggested 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.
TL;DR: By computer analyses, two different putative insulin response elements (IREs) are identified in the ME gene promoter, hereafter referred to as IRE-I (-683 to -692), which is similar to the phosphoenol pyruvate carboxy kinase promoter IRE and IRA-II (-161 to -170, which isSimilar to the glyceraldehyde phosphate dehydrogenase gene promoter I RE-A.
Abstract: Diabetes in rats is characterized by insulin deficiency accompanied by a decrease in lipogenic enzymes. The malic enzyme (ME) gene, which encodes an important lipogenic enzyme, was used to investigate insulin regulation of gene expression. ME mRNA levels were reduced by more than 90% in the liver of diabetic rats. The administration of insulin (3 U/15 days) to either control or diabetic rats increased ME mRNA by 2- to 10-fold, respectively. Since diabetes reduces circulating T3 and the levels of nuclear T3-receptors, the potential role of thyroid hormone on insulin regulation of ME gene expression was also evaluated in thyroidectomized-diabetic rats. In these animals the levels of ME mRNA were undetectable but were increased by insulin even in the absence of thyroid hormones. These in vivo effects of insulin and T3 were not additive. The transcription rate of the gene was also reduced in the diabetic liver and recovered after insulin therapy. By computer analyses we have identified two different putative insulin response elements (IREs) in the ME gene promoter, hereafter referred to as IRE-I (-683 to -692), which is similar to the phosphoenol pyruvate carboxy kinase promoter IRE and IRE-II (-161 to -170), which is similar to the glyceraldehyde phosphate dehydrogenase gene promoter IRE-A. Results from gel retardation assays suggest that a single nuclear protein binds to IRE-I whereas two different nuclear proteins bind to IRE-II. The protein/IRE-I complex increased in liver nuclear extracts from diabetic rats and decreased after insulin administration.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Water-shell and induced-fit models for the enzyme in AOT/isooctane reverse micelles at large and small [H2O]/[AOT] ratios to explain this data, respectively are proposed.
Abstract: Pigeon-liver malic enzyme has a tendency to aggregate at a large concentration of protein. The larger aggregates (hexamer and octamer) were demonstrated to be enzymically active with specific activity similar to that of the tetramer. When the enzyme was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl)-sulfosuccinate (AOT) in isooctane, the tetrameric enzyme dissociated into monomers. The dissociated monomers were also enzymically active but with diminished specific activity relative to the activity in aqueous media. The decreased enzyme activity in reverse micelles was due to interactions of surfactant with the enzyme molecules, suggesting that the cytosolic malic enzyme is located near the plasma membrane. When the dissociation was monitored by altering the degree of hydration of the system (represented by the ratio [H2O]/[AOT]), the detergent and organic solvent slightly affected KTD, the dissociation constant of tetramer to dimers (T 2 D), but increased KDM, the dissociation constant of dimer to monomers (D 2 M), by 1-2 orders of magnitude; this change caused a 2-3 orders of magnitude increase in the overall dissociation constant KTM (T 4 M). The dissociation of the tetrameric malic enzyme to monomers was favored by approximately 16 kJ/mol in AOT/isooctane reverse micelles versus aqueous media. We propose water-shell and induced-fit models for the enzyme in AOT/isooctane reverse micelles at large and small [H2O]/[AOT] ratios to explain this data, respectively. The asymmetric quaternary structure of the enzyme [Lee, H. J. & Chang, G. G. (1990) FEBS Lett. 277, 175-179] was re-evaluated in terms of the subunit interactions and various interconvertible enzyme forms.
TL;DR: The calculated values of the intrinsic 13C and deuterium isotope effects with NADP are similar to the previously determined values for the chicken liver malic enzyme and suggest that the transition-state structures are similar for the Ascaris NAD- and chicken liver NADP-malic enzymes.
Abstract: The NAD-malic enzyme catalyzes the divalent metal-ion-dependent oxidative decarboxylation of L-malate to yield CO2, pyruvate, and the reduced dinucleotide. With Mg2+ as the divalent metal ion activator, primary deuterium and tritium isotope effects have been obtained with several different alternative dinucleotide substrates. The partitioning ratio of oxalacetate to malate and pyruvate has also been determined with either NAD or 3-acetylpyridine adenine dinucleotide (3-APAD). These data have been used to calculate estimates of commitment factors and intrinsic isotope effects for the NAD-malic enzyme reaction. The calculated values of the intrinsic 13C and deuterium isotope effects with NADP are similar to the previously determined values for the chicken liver malic enzyme (Grissom, C.B., & Cleland, W. W. (1988) Biochemistry 27, 2927) and suggest that the transition-state structures are similar for the Ascaris NAD- and chicken liver NADP-malic enzymes. With NAD or NADP as the dinucleotide substrate, the data are all consistent with a stepwise chemical mechanism with oxidation of L-malate at C2 preceding decarboxylation of the bound oxalacetate intermediate. However, none of the data with the alternative dinucleotide substrates, 3-acetylpyridine adenine dinucleotide and 3-pyridine aldehyde adenine dinucleotide (PAAD), can be fit with satisfaction to the various criteria that support a stepwise mechanism with NAD(P). The mechanism with 3-APAD and PAAD is likely concerted. The most likely explanation for a change in the mechanism for oxidative decarboxylation from stepwise with NAD(P) to concerted with alternative dinucleotide substrates such as 3-APAD and PAAD is a difference in the configuration of bound malate when the different dinucleotide substrates are used.
TL;DR: The results indicate that zinc, vanadate and selenate are not insulin-mimetics under all conditions, and their effects on other insulin-sensitive processes may be fortuitous and unrelated to actions or components of the insulin signalling pathway.
Abstract: Insulin regulates the expression of genes involved in a variety of metabolic processes. In chick-embryo hepatocytes in culture, insulin amplifies the tri-iodothyronine (T3)-induced enzyme activity, and the level and rate of transcription of mRNA for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone, however, has little or no effect on the expression of these genes. In chick-embryo hepatocytes, the mechanism by which insulin regulates the expression of these or other genes is not known. Several recent studies have compared the effects of zinc, vanadate and selenate on insulin-sensitive processes in an attempt to probe the mechanism of insulin action. Because zinc, vanadate and selenate mimic the effects of insulin on several processes, they have been termed insulin-mimetics. We have studied the effect of zinc, vanadate and selenate on the T3-induced expression of both FAS and ME. Like insulin, these agents had little or no effect on the basal activities for FAS and ME in chick-embryo hepatocytes in culture for 48 h. Unlike insulin, however, zinc, vanadate and selenate inhibited the T3-induced activities and mRNA levels of both FAS and ME. Maximal inhibition was achieved at concentrations of 50 microM zinc or vanadate, or 20 microM selenate. Zinc and vanadate also inhibited the T3-induced transcription of the FAS and ME genes. Although the mechanism of this inhibition is unknown, our results indicate that it is not mediated through inhibition of binding of T3 to its nuclear receptor nor through a general toxic effect. Thus zinc, vanadate and selenate are not insulin-mimetics under all conditions, and their effects on other insulin-sensitive processes may be fortuitous and unrelated to actions or components of the insulin signalling pathway.
TL;DR: The co-ordinated nature of metabolism and the genotype-dependent differences in metabolic flux may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.
Abstract: The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of 13C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. alpha,alpha-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD+ balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in AdhF larvae. In AdhS larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic flux may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.
TL;DR: Evidence suggests that a reversible reduction and oxidation of disulfides bonds may take place during the regulation of the enzyme, indicating that the redox state of the disulfide bonds of C4 NADP-malic enzyme from maize leaves is important for the expression of maximal catalytic activity.
TL;DR: NADP- malic enzyme II, one of two isoenzymes of NADP-malic enzyme (EC 1.1.40) in Trypanosoma cruzi epimastigotes, presents hysteretic behavior that results in a kinetic lag in the reaction progress curve that suggests that hysteresis is due to an association-dissociation process influenced by the binding of these ligands to the enzyme.
TL;DR: Data and the different ratios of citrate synthase to mitochondrial malic enzyme activities found in different regions of brain suggest that human brain mitochondria, like the mitochondria isolated from other mammalian brains, are extremely heterogenous.
TL;DR: The role of dry matter, malic acid, pectinesterase and certain enzymes of gluconeogenic and organic acids metabolism in uneven ripening is discussed in this paper.
Abstract: Physico-chemical changes, organic acids content and activities of enzymes related to softening, glycolytic, gluconeogenic and organic acids metabolism were studied from 70 to 100 days after anthesis in ripening of berries of Gulabi grape. Uneven ripening was observed in 70 day old clusters. The disorder affected 22. 76 and 32.38% of berries in bunches harvested at 90 and 100 days, respectively. Unripe berries had less dry matter, sugars, skin anthocyanins and more seed, fresh weight and acidity compared to ripe berries. Green berries had more malic and less tartaric acid, while brown berries had less malic acid and black berries contained more tartaric acid. Green berries contained more polygalacturonase, cellulase, hexokinase, glucose 6-phosphatase, phosphoenol pyruvate carboxylase, pyruvate decarboxylase and polyphenol oxidase activities than ripe fruit. Brown berries contained the highest phosphofructokinase, malate and glutamate dehydrogenase activities, and black berries had the highest activities of pectinesterase, fructose 1,6-bisphosphatase and malic enzyme. The role of dry matter, malic acid, pectinesterase and certain enzymes of gluconeogenic and organic acids metabolism in uneven ripening is discussed.
TL;DR: Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus, and the phenotypes are unlinked to polymorphisms forsMDH-3,4* andmMEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.
Abstract: Electrophoretic studies of malate oxidoreductases routinely assess variation in two enzymes, malate dehydrogenase (EC 1.1.1.37) and malic enzyme (NADP+) (EC 1.1.1.40). By modification of the standard isozyme staining conditions for these enzymes, we have resolved a new NAD+-preferring, MgCl2-requiring malic enzyme which is indicated to be EC 1.1.1.39. The enzyme was detected in 10 salmonid fish species of the generaSalmo, Salvelinus, andOnchoryhncus. Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus. Inheritance inS. salar follows a single-locus model and the phenotypes are unlinked to polymorphisms forsMDH-3,4* andmMEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.
TL;DR: Findings of developmental differences in malic enzyme activity suggest that alternate substrates such as anaplerotic amino acids may be used in the young animal as energy substrates by way of the tricarboxylic acid cycle.
Abstract: Using a sensitive technique measuring 14CO2 production from radiolabeled malate, we examined malic enzyme activity in both adult and newborn rat lung tissue and in L2 cells, a cell culture line of type II pneumocytes. Malic enzyme was present in both cytosolic and mitochondrial fractions. Time course experiments demonstrated a linear rate after the initial 10 min, up to 30 min. The optimal pH in the cytosolic fraction was 8.0, whereas maximal mitochondrial malic enzyme activity occurred at pII 7.0. The mitochondrial fraction exhibited biphasic kinetics over the 200-fold range of concentrations examined. The high-affinity Km was 0.16 mmol with Vmax of 7.11 nmol/mg protein/min. The low-affinity Km was 6.95 mmol, with Vmax of 31.82 nmol/mg protein/min. In the cytosol there was a single Km of 0.30 mmol and Vmax of 5.95 nmol/mg protein/min. In paired experiments examining differences between 1-d-old and adult rat lung, significantly higher total and mitochondrial malic enzyme activity-occurred in the newborn as compared with the adult. Malic enzyme activity was also present in the L2 cells. The finding of malic enzyme activity in the lung suggests that cytosolic malic enzyme may play a role in generating NADPH needed in the lung for fatty acid synthesis. These findings of developmental differences in malic enzyme activity suggest that alternate substrates such as anaplerotic amino acids may be used in the young animal as energy substrates by way of the tricarboxylic acid cycle.
01 Jan 1994
TL;DR: In vivo the rate of delivery of malate to the bacteroid must be carefully regulated to optimise N2 fixation, and the relative rates of poly-β-hydroxybutyrate synthesis and tricarboxylic acid cycle operation are interpreted.
Abstract: Recent measurements of respiration and nitrogen fixation by isolated soybean bacteroids, incubated with oxyleghaemoglobin in a flow chamber under steady-state conditions, have shown that while addition of malate stimulated respiration, the effect on N2 fixation depended on the concentration of malate and oxygen. At low malate concentrations, N2 fixation was stimulated, but at higher malate concentrations (more than 0.5 mM at less than 60 nM O2) N2 fixation was inhibited and carbon diverted to poly-β-hydroxybutyrate formation. These results are interpreted in terms of the redox poise of pyridine nucleotides and the relative rates of poly-β-hydroxybutyrate synthesis and tricarboxylic acid cycle operation. Soybean bacteroids contain both NAD- and NADP-linked malic enzymes which have very different affinities for malate, and thereby have the capacity to alter the NAD(P)H/NAD(P) ratios in the bacteroid in response to varying malate concentrations. It is suggested that in vivo the rate of delivery of malate to the bacteroid must be carefully regulated to optimise N2 fixation.
Journal Article•
TL;DR: Pigeon liver malic enzyme is a homotetramer composed of four chemically identical subunits that causes biphasic conformational changes, the enzyme tetramers being dissociated to monomers in phase I transition.
Abstract: Pigeon liver malic enzyme (EC 1.1.1.40) is a homotetramer composed of four chemically identical subunits. Exposure of the enzyme in guanidinium chloride causes biphasic conformational changes, the enzyme tetramers being dissociated to monomers in phase I transition. Enzymic activity was completely lost in this phase. Recovery of enzyme activity was only possible at an early stage of phase I transition. Phase II is due to the enzyme unfolding, as judged by c.d. and fluorescence parameters. The steps in the transformation of native malic enzyme to a completely denatured state are in the following sequence: tetramer→ monomer→random coil
Journal Article•
TL;DR: Most of the compounds, which have been synthesized by the Central Drug Research Institute, Lucknow (India) and found to possess some anthelmintic properties, strongly inhibited the above enzymes except malic enzyme.
Abstract: The enzymes involved in the catabolism of malate namely fumarate reductase, NADH oxidase, "malic" enzyme, succinate dehydrogenase and fumarase as well as NADPH:NAD transhydrogenase, which is involved in the electron transport chain, were studied in Hymenolepis diminuta, a rat intestinal tapeworm. Among cations, K+ had no effect on any enzyme whereas Ca2+ and Mg2+ showed an increase or decrease of varying degrees of different enzyme activities. Most of the compounds, which have been synthesized by the Central Drug Research Institute, Lucknow (India) and found to possess some anthelmintic properties, strongly inhibited the above enzymes except malic enzyme.
TL;DR: It is concluded that an increased demand for NADPH in selenium deficiency, possibly resulting from increased glutathione metabolism, may induce NADPH-generating enzymes, especially malic enzyme.
Abstract: The activity of cytosolic malic enzyme was significantly higher (61%) in the liver of rats fed a selenium-deficient diet compared with the controls. Activities of two other NADPH-generating enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were also increased but to a lesser extent (13%). Glutathione reductase, an NADPH-consuming enzyme involved in the metabolism of glutathione, was higher (26%) in the liver of selenium deficient rats, possibly as a result of an increase in glutathione synthesis and enzymic processes that maintain glutathione in the reduced state. It is concluded that an increased demand for NADPH in selenium deficiency, possibly resulting from increased glutathione metabolism, may induce NADPH-generating enzymes, especially malic enzyme. ( J. Nutr. Biochem. 5:314–316, 1994 .)
TL;DR: 13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria and demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate.
Abstract: 13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron-transport system responsible for steroid hydroxylation.
[13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin.
The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.
01 Jan 1994
TL;DR: Results demonstrated that the culture on EHS-gel was better than the usual monolayer culture for investigating the effect of xenobiotics.
Abstract: Rat hepatocytes in primary culture have been used for studying growth and differentiation functions such as drug metabolism. In order to develop a culture system keeping high capacity of xenobiotics-responsiveness, effects of extracellular matrix (ECM) on gene expressions for cytochrome P-450 (P-450), 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) and malic enzyme were investigated. The treatment of xenobiotics to animals induce HMG-CoA reductase and malic enzyme as well as cytochrome P-450s. The inductions of the expression of HMG-CoA reductase gene and malic enzyme gene by polychlorinated biphenyls (PCB) were observed in primary cultured rat hepatocytes. This showed that the effect of PCB on the gene expression relating lipid metabolism was direct to hepatocytes. The induction was observed only in hepatocytes cultured on Engelbreth-Holm-Swarm sarcoma basement membrane gel (EHS-gel), not on type I collagen (TIC), which is usually used for monolayer culture of hepatocytes. The induction of CYP2B1/2 (cytochrome P-450b/e) gene expression was also observed only in the cells cultured on EHS-gel. These results demonstrated that the culture on EHS-gel was better than the usual monolayer culture for investigating the effect of xenobiotics.