Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase

Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of approximately 300 kDa compared to approximately 187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.)

The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences.     

Mitochondrial enzymes in mango fruit during ripening

Mitochondria isolated from immature (developing), mature (unripe), and ripe mango pulp actively oxidized the intermediates of the Krebs cycle. The oxidation of citrate, oxoglutarate, succinate and malate by both unripe and ripe fruit mitochondria was several fold greater than that by mitochondria from immature fruit. The levels of malic dehydrogenase and succinic dehydrogenase increased with the onset of ripening, whereas the level of citrate synthase increased several fold on maturation but decreased six-fold on ripening. Isocitrate dehydrogenase and malic enzyme were very high in the immature fruit but after a sudden decrease in the matured fruit showed a considerable rise thereafter. The ratio of the activities of isocitrate lyase to isocitrate dehydrogenase is considerably higher in the immature fruit and greatest in the unripe (mature) fruit. This, together with a higher concentration of glyoxylate at these stages, indicate the operation of the glyoxylate bypass. Oxidized and reduced forms of pyridine nucleotides were estimated.     

Fermentation and malate metabolism in response to elevated CO2 concentrations in two strawberry cultivars

Concentrations of acetaldehyde, ethanol, ethyl acetate (EA), organic acids and activities and gene expression of alcohol dehydrogenase (ADH; EC 1.1.1.1), pyruvate decarboxylase (PDC; EC 4.1.1.1), alcohol acyltransferase (AAT; EC 1.4.1.14), malate dehydrogenase (MDH; EC 1.1.1.37), malic enzyme (ME; EC 1.1.1.40) and glutamate dehydrogenase (EC 1.4.1.14) were investigated in two strawberry ( Fragaria × ananassa Duch) cultivars with different responses to CO₂ during storage. 'Jewel' fruit treated with CO₂ accumulated acetaldehyde and ethanol but little EA, while 'Cavendish' accumulated little acetaldehyde or ethanol but accumulated EA. In CO₂-treated fruit, PDC activity was positively correlated with EA accumulation in 'Jewel' but not in 'Cavendish', while no differential effect of atmosphere was observed on its gene expression. ADH activity and gene expression show a correlation with ethanol accumulation in 'Cavendish'. In 'Jewel', there was a positive correlation between ADH gene expression and enzyme activity; however, this correlation does not explain ethanol accumulation in this cultivar. EA accumulation did not show any correlation with AAT activity and gene expression in any of the cultivars. Succinate concentrations were highest and those of malate lowest in CO₂-treated fruit of both cultivars, but MDH and ME activities were not affected by CO₂. Gene expression of MDH and ME were not affected by atmosphere in 'Cavendish', although in 'Jewel' the MDH expression was slightly lower in CO₂- than air-treated fruit. The results of this study show that differences in fermentation products and malate accumulation in CO₂-treated strawberry fruit are not consistently correlated with enzyme activities and gene expression.     

Physiology and metabolism of two alkane oxidizing and citric acid producing strains of Candida parapsilosis

Summary The activity of enzymes of the tricarboxylic acid (TAC) and glyoxylate (GC) cycles in Candida parapsilosis (wild type KSh 21 and mutant 337) were studied under different physiological and metabolic conditions. C. parapsilosis differed in most of its enzyme activities from other non-citric acid producing yeasts. Furthermore, pH-value, temperature and age of culture proved to act differently on both strains of the tested organism.The addition of trans-aconitate increased not only the growth but also the activities of citrate synthase and some other enzymes while that of aconitase decreased enormously.The high citrate synthase activity might be connected with the role of citrate in the transport of acetyl groups.Abbreviations CS citrate synthase - AC aconitase - ICDH isocitrate dehydrogenase - GDH glutamate dehydrogenase - Fum fumarase - MDH malate dehydrogenase - ICL isocitrate lyase - MS malate synthase     

Adaptation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to Al-Citrate: Involvement of Tricarboxylic Acid and Glyoxylate Cycle Enzymes and the Influence of Phosphate

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by ¹H NMR and ¹³C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.     

Poly-β-hydroxybutyrate (PHB) biosynthesis,tricarboxylic acid activity and PHB content in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) root nodule

An experiment was conducted to assess the relationship between poly-β-hydroxybutyrate (PHB) biosynthesis and tricarboxylic acid (TCA) activity in desi and kabuli chickpea (Cicer arietinum L.) genotypes. The specific activities of enzymes of PHB metabolism viz., β-ketothiolase (PHB-A), acetoacetyl coenzyme A reductase (PHB-B) and PHB synthase (PHB-C), and those of tricarboxylic acid cycle (citrate synthase (CS) and malate dehydrogenase (MDH) under symbiosis were measured in bacteroids and compared with the PHB accumulation in the nodule and the root. The significant positive correlation was observed between shoot and nodule mass and PHB-A, PHB-B, and PHB-C activities. However, nodule and shoot weights were not significantly correlated with PHB content either in the roots or nodules. The same was true for PHB levels and citrate synthase activity. MDH activity showed a significant negative correlation with nodule PHB. A marked variation and an age dependant increase in malate dehydrogenase activity were measured. A higher capacity for malate oxidation by an increased MDH is likely alter the balance between malate decarboxylation and oxidation, resulting in a higher steady-state concentration of oxaloacetate and that may favor the utilization of acetyl-CoA in the TCA cycle rather than for the synthesis of PHB.     

The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus)

Exudation of organic anions is believed to be a common tolerance mechanism for both aluminium toxicity and phosphorus deficiency. Nevertheless, which of these stresses that actually elicit the exudation of organic anions from rape ( Brassica napus L) remains unknown, and the combined effects of Al toxicity and P deficiency on rape have not been reported before. Therefore, in the current study, Brassica napus var. Natane nourin plants grown with or without 0.25 m M P were exposed to 0 or 50 µ M AlCl₃ and several parameters related to the exudation of organic anions from the roots were investigated. Eight days of P deficiency resulted in a significant growth reduction, but P deficiency alone did not induce exudation of organic anions. In contrast, Al strongly induced organic acid exudation, while simultaneously inhibiting root growth. Increased in-vitro activity of citrate synthase (CS, EC 4.1.3.7), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), together with reduced root respiration, indicated that the Al-induced accumulation and subsequent exudation of citrate and malate were associated with both increased biosynthesis and reduced metabolism of citric and malic acid. Phosphorus-sufficient plants showed more pronounced aluminium-induced accumulation and exudation of organic anions than P-deficient plants. A divided root chamber experiment showed the necessity of direct contact between Al and roots to elicit exudation of organic anions. Prolonged exposure (10 days) to Al resulted in a decrease in the net exudation of citrate and malate, and the rate of decrease was much more rapid in P-deficient plants than in P-sufficient plants. It is concluded that P nutrition affects the level of Al-induced synthesis and exudation of organic anions. However, the mechanism needs further investigation.     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

Comparative studies of coupled assays for phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable K_m values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the V_max value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, V_max is brought back to the same level as that with the NADH-coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose-6-phosphate in concentrations higher than 1 m M blocks the response of PEPC to added MDH in the CS assay.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Isolation and characterization of six peach cDNAs encoding key proteins in organic acid metabolism and solute accumulation: involvement in regulating peach fruit acidity

As in many other fleshy fruits, the predominant organic acids in ripe peach ( Prunus persica (L.) Batsch) fruit are malic and citric acids. The accumulation of these metabolites in fruit flesh is regulated during fruit development. Six peach fruit-related genes implicated in organic acid metabolism (mitochondrial citrate synthase; cytosolic NAD-dependent malate dehydrogenase, and cytosolic NADP-dependent isocitrate dehydrogenase) and storage (vacuolar proton translocating pumps: one vacuolar H⁺-ATPase, and two vacuolar H⁺-pyrophosphatases) were cloned. Five of these peach genes were homologous to genes isolated from fruit in other fleshy fruit species. Phylogenetic and expression analyses suggested the existence of a particular vacuolar pyrophosphatase highly expressed in fruit. The sixth gene was the first cytosolic NAD-dependent malate dehydrogenase gene isolated from fruit. Gene expression was studied during the fruit development of two peach cultivars, a normal-acid (Fantasia) and a low-acid (Jalousia) cultivar. The overall expression patterns of the organic acid-related genes appeared strikingly similar for the two cultivars. The genes involved in organic acid metabolism showed a stronger expression in ripening fruit than during the earlier phases of development, but their expression patterns were not necessarily correlated with the changes in organic acid contents. The tonoplast proton pumps showed a biphasic expression pattern more consistent with the patterns of organic acid accumulation, and the tonoplast pyrophosphatases were more highly expressed in the fruit of the low-acid cultivar during the second rapid growth phase of the fruit.     

A novel metabolic network leads to enhanced citrate biogenesis in <Emphasis Type="Italic">Pseudomonas </Emphasis><Emphasis Type="Italic">fluorescens</Emphasis> exposed to aluminum toxicity

Aluminum (Al), an environmental toxin, is known to have a negative impact on various biological systems. However, some microbes have devised intricate mechanisms to combat the toxic influence of this trivalent metal. In this study, Pseudomonas fluorescens grown in malate invoked a unique metabolic shift to promote the synthesis of citrate, a metabolite involved in the sequestration of Al. Electrophoretic and spectrophotometric assays revealed several malate-metabolizing enzymes including malate dehydrogenase (MDH) and malic enzyme (ME) displayed increases in activity and expression in the Al-treated cells. Whereas pyruvate dehydrogenase (PDH) also showed increased activity and expression in the Al-stressed cultures, phosphoenolpyruvate carboxykinase (PEPCK) displayed a marked diminution in the Al-treated cells. The upregulation of citrate synthase (CS) coupled with the diminished activities of aconitase (ACN) and NAD-isocitrate dehydrogenase (NAD-ICDH) appeared to be instrumental in the accumulation of citrate. HPLC experiments revealed high levels of citrate in the Al-stressed cultures. Thus, an Al-enriched environment provoked a metabolic shift in P. fluorescens dedicated to the conversion of malate to citrate.     

Effect of etiroxate on LCAT activity and on certain energy metabolism enzymes in the rat

Etiroxate (Skleronorm Grünenthal R) was administered 42 days to male Wistar rats and their serum and liver cholesterol and triglyceride levels, the rate of esterification of free cholesterol in their plasma by lecithin cholesterol acyltransferase (LCAT) (EC 2.3.1.43) and thriosephosphate dehydrogenase (TPDH) (EC 1.2.1.12), lactate dehydrogenase (LDH) (EC 1.1.1.27), hexokinase (HK) (EC 2.7.1.1), c-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), malate dehydrogenase (MDH) (EC 1.1.1.37) citrate synthase (CS) (EC 4.1.3.7) and hydroxyacylcoenzyme A dehydrogenase (HOADH) (EC 1.1.1.35) activity were determined in their liver. After 14 and 28 days, animals given etiroxate (600 micrograms/kg) had smaller weight increments than the controls and a significantly lower plasma free and esterified cholesterol level, but a significantly higher liver cholesterol concentration. Their final plasma and liver cholesterol concentrations did not differ significantly from the control values. Plasma triglyceride levels were significantly raised in treated animals at all the given intervals. LCAT activity was significantly higher throughout the whole time of treatment, with the maximum increase in the last phase. Glycolytic and oxidative enzyme activities were significantly raised, whereas GPDH activity was the same as in the controls. The results show that etiroxate accelerates cholesterol turnover in the endogenous pool by activating LCAT and stimulating energy metabolism.     

Mitochondrial malate dehydrogenases in Brassica napus: altered protein patterns in different nuclear mitochondrial combinations

Two-dimensional analyses of mitochondrial proteins of Brassica napus revealed a set of differences in patterns of mitochondrial matrix proteins isolated from different nuclear backgrounds. One of these varying proteins was identified as mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) by homology analyses of the partial amino acid sequence. Immunological detection identified additional mMDH subunits and detected different patterns of mMDH subunits in two distinct mitochondria types although they were isolated from plants with the same nuclear genotype. These differences are also reflected in isozym patterns, whereas Southern analyses showed no alteration in genome structure. Therefore mitochondria type-specific mMDH modifications are possible.     

Interaction between citrate synthase and mitochondrial malate dehydrogenase in the presence of polyethylene glycol

Pig heart citrate synthase and mitochondrial malate dehydrogenase interact in polyethylene glycol solutions as indicated by increased solution turbidity. A large percentage of both enzymes sediments when mixtures of the two in polyethylene glycol are centrifuged, whereas little if any of either enzyme sediments in the absence of the other. The observed interaction is highly specific in that neither cytosolic malate dehydrogenase nor nine other proteins showed evidence of specific interaction with either pig heart citrate synthase or mitochondrial malate dehydrogenase. Escherichia coli citrate synthase did not interact with pig heart citrate synthase, but did show evidence of interaction with pig heart mitochondrial malate dehydrogenase. The relation between enzyme behavior in polyethylene glycol solution and in the mitochondrion and the significance of possible in vivo interactions between citrate synthase and mitochondrial malate dehydrogenase are discussed.     

Changes induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leaves

The effects of iron deficiency and iron resupply on the metabolism of leaf organic acids have been investigated in hydroponically grown sugar beet. Organic acid concentrations and activities in leaf extracts of several enzymes related to organic acid metabolism were measured. Enzymes assayed included phosphoenol pyruvate carboxylase (PEPC; EC 4.1.1.31), different Krebs cycle enzymes: malate dehydrogenase (MDH; EC 1.1.1.37), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2), citrate synthase (CS; EC 4.1.3.7) and isocitrate dehydrogenase (ICDH; EC 1.1.1.42), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and two enzymes related to anaerobic metabolism (lactate dehydrogenase [LDH]; EC 1.1.1.27, and pyruvate decarboxylase [PDC]; EC 4.1.1.1). Iron concentration in leaves was severely decreased by iron deficiency. Iron resupply caused an increase in iron concentrations, reaching levels similar to the controls in 96 h. Iron deficiency induced a 2.3-fold (from 16 to 37 mmol m⁻²) increase in leaf total organic acid concentration. Organic anion concentrations were still 4-fold higher than the controls 24 h after resupply and decreased to values similar to those found in the controls after 96 h. All measured enzymes had increased activities in extracts of iron-deficient leaves when compared to the controls and generally decreased to control values 24 h after iron addition. These data provide evidence that organic acid accumulation in iron-deficient leaves is likely not due to an enhancement in leaf carbon fixation. Instead, this accumulation could be associated with organic acid export from the roots to the leaves via xylem.     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide