Concentration Of L-malate Research Articles

Metabolic engineering of Escherichia coli for l-malate production anaerobically

Backgroundl-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce l-malate from renewable resources has attracted significant attention.ResultsA potential l-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in l-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final l-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied.ConclusionsThe availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of l-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of l-malate.

The impact of hybrid yeasts on the aroma profile of cool climate Riesling wines

The current study highlights the effects of intra- and interspecific hybrid yeasts of the genus Saccharomyces (S.) on the alcoholic fermentation and formation of aroma compounds in cool climate Riesling wines. Three different hybrid yeasts: S. cerevisiae × S. paradoxus (SC × SP), S. cerevisiae × S. kudriavzevii (SC × SK) and S. cerevisiae var. cerevisiae × S. cerevisiae var. bayanus (SC × SB) were investigated. The species S. cerevisiae var. bayanus (SB) was chosen as control variant. It has been demonstrated that the hybrid yeasts have the ability to preserve positive properties while, suppressing undesired properties from the parental yeast species. The hybrid SC × SK showed an increase of desired acetate esters and monoterpenes. The concentrations of higher alcohols were higher in wines fermented by SC × SP, compared to the other variants. SC × SP fermentations resulted in decreased concentrations of l-malate and sulphites.

Possible involvement of phosphoenolpyruvate carboxylase and NAD-malic enzyme in response to drought stress. A case study: a succulent nature of the C4-NAD-ME type desert plant, Salsola lanata (Chenopodiaceae).

The co-ordination between the primary carboxylating enzyme phosphoenolpyruvate carboxylase (PEPC) and the further decarboxylating enzymes is crucial to the efficiency of the CO2-concentrating mechanism in C4 plants, and investigations on more types of C4 plants are needed to fully understand their adaptation mechanisms. In this study we investigated the effect of drought on carboxylating enzyme PEPC, and the further decarboxylating NAD-malic enzyme (NAD-ME) of Salsola lanata Pall. (Chenopodiaceae) - an annual succulent C4-NAD-ME subtype desert plant. We investigated enzyme activity at the transcriptional level with real-time quantitative PCR and at the translational level by immunochemical methods, and compared S. lanata with other forms of studied C4 plants under drought stress. Results showed that only severe stress limited PEPC enzyme activity (at pH 8.0) of S. lanata significantly. Considering that PEPC enzyme activity (at pH 8.0) was not significantly affected by phosphorylation, the decrease of PEPC enzyme activity (at pH 8.0) of S. lanata under severe stress may be related with decreased PEPC mRNA. The suggestion of increased phosphorylation of the PEPC enzyme in plants under moderate stress was supported by the ratio of PEPC enzyme activity at pH 7.3/8.0, as PEPC enzyme is inhibited by L-malate and the evidence of the 50% inhibiting concentration of L-malate. NAD-ME activity decreased significantly under moderate and severe stress, and coincided with a change of leaf water content rather than the amount of α-NAD-ME mRNA and protein. Leaf dehydration may cause the decrease of NAD-ME activity under water stress. Compared with other C4 plants, the activities of PEPC and NAD-ME of S. lanata under drought stress showed distinct features.

Robust l-malate bienzymatic biosensor to enable the on-site monitoring of malolactic fermentation of red wines

Monitoring the malolactic fermentation process is strictly required to guarantee the sensorial quality and freshness of red wines. This could be achieved by in-field and real-time continuous measurements of l-malate concentration in the fermentation tanks. The potential of a miniaturized amperometric bienzymatic biosensor as an analytical tool to be applied in such scenario is described in this paper. The biosensor comprises a thin-film gold electrode as transducer, malate dehydrogenase (MDH) and diaphorase (DP) enzymes together with nicotinamide adenine dinucleotide (NAD+) cofactor as the selective receptor and an adequate redox mediator to record the corresponding amperometric signal. Three different biosensor architectures are studied, whose main differences lie in the immobilization of the different chemical components onto the electrode surface. In all cases a fast-electrosynthethized polypyrrole (PPy) membrane is generated for this purpose. The experimental conditions are optimized and the best architecture shows a sensitivity of 1365 ± 110 mA M−1 cm−2 and a detection limit of 6.3 × 10−8 M in a concentration range of 1 × 10−7 M – 1 × 10−6 M. The biosensor presents an excellent working stability as it retains above 90% of its sensitivity after 37 days, thus enabling the monitoring of the malolactic fermentation of three red wines. The obtained results show excellent agreement with the standard colorimetric method.

Methylene Green Electrodeposited on SWNTs-Based “Bucky” Papers for NADH and l-Malate Oxidation

This research introduces a cavity anode design based on new single-walled nanotube (SWNTs) papers, "bucky" papers, used for the oxidation (and regeneration) of nicotinamide adenine dinucleotide (NADH) and the oxidation of l-malate. The materials designed are paper-like processed composites containing also additives: BP11 sample contains SWNTs and isopropanol (IPA); the BPMG sample contains SWNTs, IPA, and methylene green (MG). NADH/NAD(+) is the cofactor responsible for the oxidation of l-malate by malate dehydrogenase (MDH), in the Krebs' cycle. Because of the high overpotential of NADH oxidation, poly methylene green (PMG) was utilized as the electrocatalyst to produce NAD(+). The electrocatalyst was deposited on the surface of the "bucky" papers by electropolymerization by means of 10 voltammetric cycles in a range of -0.5 V and +1.3 V (vs Ag/AgCl reference electrode) at a scan rate of 5 mV/s. The catalytic performance of PMG was evaluated by chronoamperometric measurements of NADH oxidation at 0.3 V in phosphate buffer and l-malate oxidation at 0.1 V in the presence of MDH. For both "bucky" papers, the chronoamperometric curves of PMG, current vs NADH concentration, show a linear relationship demonstrating to have a first order Fick's law behavior for concentrations of NADH lower than 6 mM. The chronoamperometric curves in the presence of MDH, current against l-malate concentration, show a Michaelis-Menten behavior where no inhibition or competitive reaction are detected. Additionally, the anodic materials were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS), the polymerization of MG is effectively observed in the form of particles nucleation. The anodes show an excellent electrocatalytic activity toward NADH oxidation. The electrode design is feasible, reproducible, and overall stable.

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4‐like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).

An effective cold inducible expression system developed in Pseudoalteromonas haloplanktis TAC125

A regulative two-component system previously identified in Pseudoalteromonas haloplanktis TAC125 was used to construct a cold inducible expression system that is under the control of l-malate. Performances of the inducible system were tested for both psychrophilic and mesophilic protein production using two "difficult" proteins as control. The results obtained demonstrated that both psychrophilic beta-galactosidase and yeast alpha-glucosidase are produced in a fully soluble and catalytically competent form. Optimal conditions for protein production, including growth temperature, growth medium and l-malate concentration were also investigated. Under optimized conditions yields of 620 and 27 mg/l were obtained for beta-galactosidase and alpha-glucosidase, respectively.

Specific features of changes in levels of endogenous respiration substrates in Saccharomyces cerevisiae cells at low temperature

The rate of endogenous respiration of Saccharomyces cerevisiae cells incubated at 0 degrees C under aerobic conditions in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30 degrees C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The initial concentration of oxaloacetate significantly decreased the activity of succinate dehydrogenase. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h. It is suggested that the increase in the L-malate level in the cells and the concurrent decrease in the oxaloacetate level in the mitochondria should be associated with a deceleration at 0 degrees C of the transport of endogenous respiration substrates from the cytosol into the mitochondria. This deceleration is likely to be caused by a high Arrhenius activation energy specific for transporters. The physiological significance of L-malate in regulation of the S. cerevisiae cell respiration is discussed.

Inhibition of ruminal microbial methane production by beta-cyclodextrin iodopropane, malate and their combination in vitro.

The objective of this study was to evaluate the effects of different concentrations of l-malate (0, 5, 10 and 20 mm), 2-iodopropane-beta-cyclodextrin complex (CD-IP) (0, 0.1, 0.2 and 0.4 mm) and a combination of malate (10 and 20 mm) plus CD-IP (0.2 and 0.4 mm) on methane production from corn starch. Ruminal fluid was collected from dairy cows, mixed with phosphate buffer (1 : 2) and incubated (30 ml) anaerobically at 38 degrees C for 6 h with or without additives. Fermentation of corn starch in the presence of malate resulted in an increase (p < 0.05) in pH of the medium, total volatile fatty acid (VFA), total gas production and molar proportion of propionate. Acetate and ammonia-N concentration were unchanged. Methane production was decreased (p < 0.05) (15.5 to 20.4%). Addition of CD-IP in corn starch resulted in an increase (p < 0.05) in total VFA and molar proportion of propionate. Acetate, pH and ammonia-N concentration of the medium were decreased (p < 0.05). Total gas production was unchanged. Methane production was decreased (p < 0.05) (25.2 to 97.1%) and hydrogen production was increased (p < 0.05). Addition of l-malate to CD-IP resulted in an increase (p < 0.05) in total VFA, total gas production and molar proportion of propionate. Acetate and ammonia-N concentration were decreased (p < 0.05). No effects were observed on medium pH. Methane production was decreased (p < 0.05) (49.5 to 97.1%). Hydrogen production was also decreased (p < 0.05) (54.5 to 64.1%) compared with those of CD-IP alone. Therefore, these additives may be used as supplements to inhibit methane production as well as to improve rumen fermentation and animal performance.

Counteractive Effects of ABA and GA3 on Extracellular and Intracellular pH and Malate in Barley Aleurone.

Barley (Hordeum vulgare L.) aleurone layers are known to constitutively acidify their surroundings, primarily by L-malic acid release (J. Mikola, M. Virtanen [1980] Plant Physiol 66: S-142). Here we demonstrate the antagonistic effects of the plant hormones gibberellic acid (GA3) and abscisic acid (ABA) on the regulation of extracellular pH (pHe) of barley aleurone layers. We observed a strong correlation between ABA-induced enhancement of extracellular acidification and an ABA-induced increase in L-malic acid release. In addition, ABA caused an increase in intracellular L-malate level. GA3 caused a slight decrease in intracellular L-malate level and was able to inhibit the ABA-induced increase in L-malate intracellular concentration and release. In addition, this ABA-induced L-malate release could be completely inhibited by GA3. The ABA-induced release of L-malic acid could not account for the total ABA-induced pHe decrease, suggesting the existence of an additional mechanism involved in the regulation of pHe. It has been reported that ABA induces an intracellular pH (pHi) increase, possibly due to the activation of plasma membrane proton pumps (R. Van der Veen, S. Heimovaara-Dijkstra, M. Wang [1992] Plant Physiol 100: 699-705). A pHi increase, such as that caused by ABA, might be correlated with the intracellular L-malate increase as suggested by the pH stat model of D.D. Davies ([1986] Physiol Plant 67: 702-706). We studied if the effects of GA3 on L-malate concentration were correlated with changes in pHi and found that GA3 caused a pHi decrease and that GA3 and ABA could interfere in the regulation of pHi. In addition, we were able to mimic the effect of both hormones on L-malate release by bringing about artifical pHi changes with the weak acid 5,5-dimethyl-2,4-oxazolidinedione and the weak base methylamine. The physiological meaning of the effects of GA3 and ABA on the regulation of both pHe and pHi during grain germination are discussed.

The Interactive Effects of pH, L-Malate, and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase.

The interactive effects of pH, L-malate, and glucose-6-phosphate (Glc-6-P) on the Vmax and Km of guard-cell (GC) phosphoenolpyruvate (PEP) carboxylase (PEPC) of Vicia faba L. were determined. Leaves of three different physiological states (closed stomata, opening stomata, open stomata) were rapidly frozen and freeze dried. GC pairs dissected from the leaves were individually extracted and individually assayed for the kinetic properties of PEPC. Vmax was 6 to 9 pmol GC pair-1 h-1 and was apparently unaffected to a biologically significant extent by the investigated physiological states of the leaf, pH (7.0 or 8.5), L-malate (0, 5, or 15 mM), and Glc-6-P (0, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the Km(PEP.Mg) was about 0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a GC [PEP] of 0.27 mM. In the study reported here, we determined that the in situ GC [Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Glc-6-P was included in the GC PEPC assay mixture, the Km(PEP.Mg) decreased to about 0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Glc-6-P at endogenous concentrations would seem both to activate the enzyme and to diminish the dramatic effect of pH on Km(PEP.Mg). Under assay conditions, L-malate is an inhibitor of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8 mM. Inclusion of 5 mM L-malate increased the Km(PEP.Mg) to about 3.6 mM (pH 7.0) or 0.4 mM (pH 8.5). Glc-6-P (0.5 mM) was sufficient to relieve L-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM Glc-6-P was required to relieve L-malate inhibition at pH 7.0. No biologically significant effect of physiological state of the tissue on GC PEPC Km(PEP.Mg) (regardless of the presence of effectors) was observed. Together, these results are consistent with a model that GC PEPC is regulated by its cytosolic chemical environment and not by posttranslational modification that is detectable at physiological levels of effectors. It is important to note, however, that we did not determine the phosphorylation status of GC PEPC directly or indirectly (by comparison of the concentration of L-malate that causes a 50% inhibition of GC PEPC).

Immobilization of the tetrameric and monomeric forms of pigeon liver malic enzyme on Sepharose beads.

Pigeon liver malic enzyme was chemically attached to Sepharose 4B-CL beads. The enzyme lost approximately 50% of its original activity when immobilization was carried out with 5 mg CNBr/ml gel. Immobilization performed at pH 8.0 or pH 4.5 resulted in the formation of matrix-bound tetramer and monomer, respectively. Matrix-bound reconstituted tetramer was derived from matrix-bound monomer by mixing the latter with soluble enzyme at pH 4.5, then raised the pH of the solution to 8.0. The matrix-bound monomer was demonstrated to be enzymically fully active in terms of specific activity. The pH profile for the enzymic reaction was similar for both soluble and immobilized enzymes. However, the latter had a broader range for the optimum pH (pH 6.8-7.8). The Arrhenius plots for all immobilized enzyme forms were biphasic with inflection at approximately 27 degrees C. The apparent Michaelis constants for the substrates increased about 2-3-fold after immobilization. All immobilized enzyme forms, including the matrix-bound monomer, showed substrate inhibition at high concentrations of L-malate. Both high-affinity and low-affinity binding sites for Mn2+ existed for all immobilized enzyme forms. These results are consistent with an existing asymmetric model, but are not compatible with a sequential model for the enzyme tetramer. The immobilized enzyme was stable for at least four months at 4 degrees C. As compared to soluble enzyme, the immobilized enzyme was less inhibited by (NH4)2SO4 or NaCl. It was also resistant to inactivation with periodate-oxidized aminopyridine adenine dinucleotide phosphate, an affinity label for malic enzyme. Incubation of the immobilized enzyme (1.25 microM) with the reagent (5.6 mM) resulted in pseudo-first-order inactivation with a rate constant of 0.0108 min-1 that was at least an order of magnitude smaller than that for the soluble enzyme.

Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate.

Mitochondrial malate dehydrogenase shows a complex regulation pattern in the presence of citrate. Previously published results indicate that this enzyme is activated by citrate in the NAD(+)----NADH direction and inhibited in the opposite direction. Moreover, high concentrations of L-malate or oxaloacetate produce deviations from the Michaelis-Menten behaviour. Results reported in this paper clearly show that citrate both activates and inhibits mitochondrial malate dehydrogenase in the same direction (NAD(+)----NADH), and in the same reaction medium, depending on substrate concentration. This surprising effect has made it necessary to propose a new kinetic mechanism that extends those previously suggested and allows us to explain both the citrate effect (activating or inhibitory) and the effect of high concentrations of L-malate and oxaloacetate.

L-Malate-sensing electrode based on malate dehydrogenase and NADH oxidase

An L-malate-sensing electrode was constructed from an oxygen electrode and a layer containing immobilized malate dehydrogenase (MDH) and NADH oxidase. MDH catalyses the dehydrogenation of L-malate by NAD + and NADH oxidase catalyses the regeneration of NAD + with the use of oxygen. The regeneration enables the L-malate oxidation to proceed efficiently even in a medium of neutral pH. At pH 8.0, L-malate in the concentration range 5 μM–1.5 mM can be measured. The relative standard deviation for the measurement is 1.2% ( L-malate concentration, 0.2 mM; n=10). The present L-malate-sensing electrode is stable for 8 weeks. A two-electrode sensor system consisting of the L-malate-sensing electrode and an L-lactate-sensing electrode based on lactate oxidase was prepared and applied to the simultaneous determination of the two components in wines.

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C(3) or C(4): CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V(max),K(m(PEP))), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4 degrees C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V(max) and increased the K(m(PEP)) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K(m(PEP)) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C(4) species studied, and are consistent with the photosynthetic metabolism of this plant.

Proposed role of lactate in germination of hypochlorite-treated Clostridium botulinum spores.

Clostridium botulinum 12885A spores treated with hypochlorite required added DL-calcium lactate for L-alanine germination. Lactate was the active component of calcium lactate. Equimolar concentrations of L-malate, but not of DL-propionate, could replace lactate, suggesting that the alpha-hydroxy acid structure is important. Neither lactate nor malate was an effective germinant for buffer-treated or hypochlorite-treated spores. If the L-alanine concentration was increased 100-fold (to 450 mM), the lactate germination requirement was overcome. The data suggest that the L-alanine germination sites were modified by hypochlorite so that a higher concentration of alanine was required for activity. Lactate appeared to be an activator of modified or non-hypochlorite-modified L-alanine germination sites.

The kinetic properties of nad-linked malic enzyme from muscle of the dung beetle Catharsius

Nad-linked malic enzyme ( L-malate: NAD + oxidoreductase (decarboxylating), E.C. 1.1.1.38) was purified from muscle of the dung beetle Catharsius to a specific activity of 28 μmol/min/mg of protein. Its molecular weight was estimated as 250,000–260,000 by Sephadex gel filtration. Activity was dependent on Mg 2+ or Mn 2+, for which K a values of 0.18 and 0.019 mM were obtained respectively. With Mg 2+, at pH 7.2, apparent K m values obtained for L-malate (with 0.76 mM NAD +) and NAD + (with 2.5 mM L-malate) were 1.40 and 0.54 mM respectively. Plots of activity versus substrate concentration tended to be sigmoidal, but became hyperbolic in the presence of succinate or fumarate. These dicarboxylic acids lowered apparent K m values, but did not affect maximum velocity. With succinate present, the apparent K m for NAD + decreased with increasing L-malate concentration towards a limiting value of 0.12 mM; the apparent K m for L-malate decreased with increasing NAD + concentration towards a limiting value of zero. The maximum velocity decreased sharply below pH 7.0; apparent K m values increased with increasing pH. With Mn 2+, plots of activity versus substrate concentration were more nearly hyperbolic and apparent K m values were lower than with Mg 2+. With NADP, maximum activities 30 and 40% those with NAD were obtained with Mg 2+ and Mn 2+ respectively. The reductive carboxylation of pyruvate occurred in the presence of Mn 2+, and with high concentrations of bicarbonate and pyruvate, at up to 5% the maximum rate of the forward reaction. A Ca 2+ stimulated oxaloacetate decarboxylase activity was associated with the enzyme.

Production of organic acids in Pisum sativum root nodules as a result of oxygen stress

Higher specific activities of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were found in root nodule tissue than in root tissue from Pisum sativum plants, inoculated with Rhizobium leguminosarum. ADH from root nodule tissue possessed a higher affinity towards acetoaldehyde than ADH from root tissue. Dynamic changes in properties and specific activities of these enzymes could be demonstrated in root tissue by restriction of aeration. Accumulation of malic acid, the first stable product of CO 2 dark fixation, resulting in a considerable concentration of L-malate in root nodules and the rapid uptake of C 4-dicarboxylic acids by isolated bacteroids are discussed in relation to the energy demand of R. leguminosarum during nitrogen fixation in symbiosis.

Hymenolepis microstoma: Lactate and malate dehydrogenases of the adult worm

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 m M phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K m values for the substrates and cofactors were as follows: pyruvate = 0.51, m M; lactate = 3.8 m M; NADH = 0.011 m M; and NAD = 0.17 m M. NADPH, NADP, or d(−)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate ( p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K m values for substrates and cofactors were as follows: l-malate = 1.09 m M; oxaloacetate = 0.0059 m M; NADH = 0.017 m M; and NAD = 0.180 m M. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 m M). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 m M and 0.5 m M, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.

Differential characteristics of the cytosol and mitochondrial isozymes of malic enzyme from bovine brain: Effects of dicarboxylic acids and sulfhydryl reagents

The cytosol and mitochondrial isozymes of bovine brain malic enzyme were studied with respect to their sensitivity towards a series of dicarboxylic acids and sulfhydryl reagents. While no effects were obtained with the dicarboxylic acids in the case of the cytosol enzyme, the activity of the mitochondrial variant was increased considerably when either succinate, 2-mercaptosuccinate, or l-aspartate were tested at low concentrations of l-malate. The activation was associated with a clear decrease in the Hill coefficient for l-malate, and this has been taken as an indication of the presence of an allosteric site on the mitochondrial enzyme. The presence of l-malate or a dicarboxylate anion analog is required at this site in order to achieve optimal velocity. The activators were also effective in increasing the reductive carboxylation of pyruvate by the mitochondrial enzyme and had no effect on the cytosol variant. The two isozymes also showed a clear differential sensitivity to 5,5′-dithiobis(2-nitrobenzoic acid) and Hg 2+, since the mitochondrial malic enzyme was inhibited by concentrations of these reagents far below those required in order to achieve an effect on the activity of the malic enzyme found in the cytosol.