Decarboxylation Of Malate Research Articles

271 EXPRESSION OF NADP-MALIC ENZYME RNA LEVELS IN RIPENING PEACH FRUIT

NADP-dependent Malic Enzyme (NADP-ME, EC 1.1.1.40) catalyzes the decarboxylation of malate, resulting in the release of CO2. In C3 plants the enzyme does not contribute CO2 directly to photosynthesis. Rather, it is associated with the supplemental synthesis of glycolytic and Krebs Cycle intermediates, although it may also be involved in regulating intracellular pH. NADP-ME activity increases during ripening of several fruits e.g. tomato and apple, usually in association with increased respiration of the developing fruit. We examined expression of NADP-ME during ripening in peach using a cDNA probe derived from F. trinervia (C4 dicot). The probe hybridized to a single RNA species of the predicted size and was low in abundance as expected for a C3 NADP-ME. As fruit matured, the RNA levels increased to a maximum around 133-140 days after bloom (fully ripe). NADP-ME RNA was not detectable from leaves isolated at the same time.

Photosynthesis in Phosphoenolpyruvate Carboxykinase-Type C4 Plants: Mechanism and Regulation of C4 Acid Decarboxylation in Bundle Sheath Cells

The mechanism and regulation of C4 acid decarboxylation in phosphoenolpyruvate (PEP) carboxykinase-type C4 plants was examined in isolated bundle sheath cell strands. These cells decarboxylated added oxaloacetate to PEP at rates exceeding 2.5 μmol min−1 mg−1 chlorophyll when ATP was added. This requirement for ATP could be replaced by malate plus ADP; ander these conditions this cytosol-located decarboxylation of oxaloacetate via PEP carboxykinase was sustained by respiratory ATP. It was confirmed that respiratory ATP production was linked primarily to the oxidative decarboxylation of malate via NAD malic enzyme. This process, measured as pyruvate production, was highly dependent on Pi. Besides being required to generate ATP, Pi had a second role which was probably associated with the transport of malate into mitochondria. Maximum rates of malate decarboxylation via NAD malic enzyme substantially exceeded the minimum rates necessary for providing ATP for cytosolic oxaloacetate decarboxylation. When malate was added with oxaloacetate, ADP and Pi rates of malate decarboxylation of between 3 and 4 μmol min−1 mg−1 chlorophyll were recorded. About half of this activity was sustained by the reoxidation of NADH coupled to reduction of oxaloacetate via malate dehydrogenase. When malate was added without oxaloacetic acid, respiration by these bundle sheath cells was stoichiometrically linked with the oxidation of nialate to pyruvate. This malate-dependent respiration was stimulated by adding ADP or phosphorylation uncouplers; it was not significantly inhibited by including oxaloacetate. Possible mechanisms of regulation of the partitioning of C4 acid decarboxylation between PEP carboxykinase in the cytosol and mitochondrial NAD malic enzyme are discussed.

The Damping and Reinitiation of the Circadian Rhythm of CO2Output inBryophyllumLeaves in Relation to their Malate Content

The circadian rhythm of CO 2 output in leaves of Bryophyllum fedtsschenkoi damps out after 3-4 d in continuous darkness and CO 2 -free air stream at 15 o C. The rhythm is reinitiated after a single exposure to white light of 2, 4, 6 or 8 h duration, damps out again after a further 3-4 d and can be reinitiated for a second time by a further exposure to light. During the exposure to light there is a burst of CO 2 output consistent with the decarboxylation of malate, and the rhythm begins afterwards with an initial high rate of CO 2 fixation. Malate gradually accumulates in the leaves in continuous darkness to attain a maximum value (35 mol m -3 ) at the time when the circadian rhythm disappears, and decreases to a low value (19 mol m -3 ) after a 4 h exposure to light which reinitiates rhythmicity

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination

Discrimination against (18)O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%(o) for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%(o)) compared with nongreen tissues (25.3-25.9%(o)). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.

Effect of Phloem-Translocated Malate on NO3− Uptake by Roots of Intact Soybean Plants

In soybean (Glycine max L. Merr. cv Kingsoy), NO(3) (-) assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO(3) (-) uptake by roots. The net NO(3) (-) uptake rate by roots of soybean plants was stimulated by the addition of K-malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO(3) (-) uptake and C excretion rates by roots. These results suggest that NO(3) (-) uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO(3) (-) uptake, and excretion of HCO(3) (-) ions was probably released by malate decarboxylation. NO(3) (-) uptake rate increased when the supply of NO(3) (-) to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO(4) (2-). We conclude that in situ, NO(3) (-) reduction rate in the shoot may control NO(3) (-) uptake rate in the roots via the translocation rate of malate in the phloem.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO 2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3 1 21 or its enantiomer, with a = b = 131.2(7) A ̊ , c = 152.6(9) A ̊ , and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (λ = 0.95 Å) to about 3.0 Å resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

A malic enzyme probe detects cross-hybridizing sequences closely linked to loci encoding other metabolic enzymes

The cytoplasmic malic enzyme ( Mod-1) catalyzes the oxidative decarboxylation of malate: malate + NADP + ⇒ pyruvate + CO 2 + NADPH + H +. Using a cDNA clone of Mod-1 as a probe, two new DNA markers not at the Mod-1 locus (restriction fragment length polymorphisms, RFLP) were detected by Southern blot analysis that showed extensive homology to Mod-1 sequences. Linkage of each restriction fragment length polymorphism to loci other than Mod-1 was assessed using the BXD ( C57BL 6J × DBA 2J ) recombinant inbred strains and confirmed by backcrosses. One polymorphic site, designated D9Rti1, was found to be closely linked to the phosphoglucomutase ( Pgm-3) locus on Chromosome 9. The other hybridization site, designated D1Rti2, was closely linked to the isocitrate dehydrogenase ( Idh-1) locus on Chromosome 1. The data presented imply that Mod-1 homologous sequences are tightly linked to three different metabolic enzymes.

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.

Intermediary carbohydrate metabolism in the adult filarial worm setaria digitata

Banu M. J., Dhandayuthapani S. and Nellaiappan K. 1991. Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitala. International Journal for Parasitology 21: 795–799. Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.

Genetic regulation of malic enzyme activity in the mouse.

Cytosolic malic enzyme catalyzes the NADP(+)-dependent oxidative decarboxylation of malate to pyruvate and CO2. Additionally, this enzyme produces large amounts of reducing equivalents (NADPH) required for de novo fatty acid synthesis and provides a precursor for oxaloacetate replacement in the mitochondria. Malic enzyme is considered a key lipogenic enzyme and changes in enzyme activity parallel changes in the lipogenic rate. As would be expected, the activity of malic enzyme responds to a variety of dietary and hormonal factors acting mainly on the rate of enzyme synthesis. In the mouse, the structural locus for malic enzyme (Mod-1) is located on chromosome 9. Two alleles reflecting differences in electrophoretic mobility have been identified. This report demonstrates that the amount of hepatic malic enzyme activity is strain-dependent and is regulated by a malic enzyme regulator locus (Mod1r) located on the proximal end of chromosome 12. Two alleles have been identified: Mod1ra, conferring high enzyme activity (C57BL/6J), and Mod1rb, conferring low enzyme activity (C57BL/KsJ). Biochemical studies have demonstrated differences in the apparent Km and Vmax and in specific activity on purification and immunoprecipitation, features that suggest changes in enzyme structure even though no differences were observed by electrophoresis and isoelectric focusing. These combined data suggest that differences in both enzyme quantity and structure may be involved in the genetic regulation of malic enzyme activity in mice.

Malic enzyme in human liver

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction. Extramitochondrial malic enzyme has been isolated from the post-mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, ADP-Sepharose-4B and Sephacryl S-300 to apparent homogeneity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 mumol.min-1.mg protein-1, which corresponds to about 10,000-fold purification. The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is a tetramer composed of identical-molecular-mass subunits. The isoelectric point of the isolated enzyme was 5.65. The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction. The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP-linked decarboxylation reaction varied with malate concentration. The Km values determined at pH 7.2 for malate and NADP were 120 microM and 9.2 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 microM and 27.9 mM, respectively. The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. The Km values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD-dependent decarboxylation reaction was not reversible. The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetate and NADPH-linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP-linked oxidative decarboxylation of malate, respectively. The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.

Mitochondrial NAD(P)-dependent malic enzyme from herring testicular tissue: Purification, kinetic behaviour and regulatory properties

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD(+) oxidoreductase (decarboxylating)] was purified from herring testicular tissue to a specific activity of 26.4 μmol NADH/min/mg protein. Herring testicular tissue is one of the most abundant sources of this enzyme. The purification procedure involved differential centrifugation of mitochondria and then chromatography on DEAE-Sephacel, Red Agarose and Sephacryl S-300. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of Mn(2+) and either NAD or NADP. Under Vmax conditions the ratios for the rate of NAD/NADP reduction was 1.8. A study of the reductive carboxulation reaction indicated that this enzyme reaction is reversible; at pH 7.0 the reverse reaction exhibited 22% of the activity of forward reaction. Some kinetic characteristics of the enzyme were determined. ATP was found to be a competitive inhibitor with respect to malate. Fumarate reversed ATP inhibition. Regulation of NAD(P)-dependent malic enzyme from herring testicular tissue mitochondria could respond to changing levels of mitochondrial ATP and fumarate in vivo.

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

The circadian rhythm of CO2 assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO2. During such exposures substantial fixation of CO2 takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40-50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO2 to normal air, a circadian rhythm of CO2 assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO2, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO2 in continuous illumination, appears to be due to the presence of a high concentration of CO2 within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.

CO2 and O2 Exchanges in the CAM Plant Ananas comosus (L.) Merr

Photosynthesis and light O(2)-uptake of the aerial portion of the CAM plant Ananas comosus (L.) merr. were studied by CO(2) and O(2) gas exchange measurements. The amount of CO(2) which was fixed during a complete day-night cycle was equal to the amount of total net O(2) evolved. This finding justifies the assumption that in each time interval of the light period, the difference between the rates of net O(2)-evolution and of net light atmospheric CO(2)-uptake give the rates of malate-decarboxylation-dependent CO(2) assimilation. Based upon this hypothesis, the following photosynthetic characteristics were observed: (a) From the onset of the light to midphase IV of CAM, the photosynthetic quotient (net O(2) evolved/net CO(2) fixed) was higher than 1. This indicates that malate-decarboxylation supplied CO(2) for the photosynthetic carbon reduction cycle during this period. (b) In phase III and early phase IV, the rate of CO(2) assimilation deduced from net O(2)-evolution was 3 times higher than the maximum rate of atmospheric CO(2)-fixation during phase IV. A conceivable explanation for this stimulation of photosynthesis is that the intracellular CO(2)-concentration was high because of malate decarboxylation. (c) During the final hours of the light period, the photosynthetic quotient decreased below 1. This may be the result of CO(2)-fixation by phosphoenolpyruvate-carboxylase activity and malate accumulation. Based upon this hypothesis, the gas exchange data indicates that at least 50% of the CO(2) fixed during the last hour of the light period was stored as malate. Light O(2)-uptake determined with (18)O(2) showed two remarkable characteristics: from the onset of the light until midphase IV the rate of O(2)-uptake increased progressively; during the following part of the light period, the rate of O(2)-uptake was 3.5 times higher than the maximum rate of CO(2)-uptake. When malate decarboxylation was reduced or suppressed after a night in a CO(2)-free atmosphere or in continuous illumination, the rate of O(2)-uptake was higher than in the control. This supports the hypothesis that the low rate of O(2)-uptake in the first part of the light period is due to the inhibition of photorespiration by increased intracellular CO(2) concentration because of malate decarboxylation. In view of the law of gas diffusion and the kinetic properties of the ribulose-1,5-bisphosphate carboxylase/oxygenase, O(2) and CO(2) gas exchange suggest that at the end of the light period the intracellular CO(2) concentration was very low. We propose that the high ratio of O(2)-uptake/CO(2)-fixation is principally caused by the stimulation of photorespiration during this period.

Mitochondrial NAD(P)-malic enzyme from herring skeletal muscle

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD(+) oxidoreductase (decarboxylating)] was purified from herring skeletal muscle to a specific activity of 8.2 μmol/min/mg. The purification procedure involved chromatography on DEAE-cellulose, Red Agarose and a Sephacryl S-300 with a final recovery of 38% of enzyme activity. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of either NAD or NADP in the presence of Mn(2+). Some kinetic characteristics of this enzyme were determined. The pH optimum of activity is 7.0. ATP was shown to be a competitive inhibitor with malate. The inhibition by ATP displayed hyperbolic competitive kinetics with a Ki (ATP) of 0.28 mM in the presence of NAD and 0.75 mM in the presence of NADP. Fumarate reversed ATP inhibition.In vivo, regulation of NAD(P)-dependent malic enzyme might respond to changing levels of mitochondrial ATP and fumarate with the enzyme undergoing kinetic activation by an increase in the concentration of mitochondrial fumarate which could reverse enzyme inhibition by ATP.

The effect of thyroid hormone on the chromatin structure and expression of the malic enzyme gene in hepatocytes.

Malic enzyme catalyzes the NADP-dependent oxidative decarboxylation of malate to pyruvate and carbon dioxide and is involved in lipogenesis. We have investigated the effect of thyroid hormone on the chromatin structure of the malic enzyme gene in rat liver. Hypersensitivity to DNase I in the immediate 5'-flanking region was altered by T3. T3 stimulation induced hypersensitive sites at -310 base pairs (bp) and -50 bp whereas a hypersensitive site at -170 bp was thyroid hormone independent. Hypersensitive sites identified in the 3'-flanking region showed no change with T3 stimulation. We further characterized expression of the malic enzyme gene as a function of thyroidal state by localizing malic enzyme mRNA in hepatocytes using in situ hybridization histochemistry. In hypothyroid and euthyroid states, two populations of hepatocytes were seen, some with malic enzyme message and others with no detectable message. These differences in malic enzyme gene expression were most evident between groups or regions of hepatocytes. After 10 days of thyroid hormone treatment all hepatocytes demonstrated malic enzyme message. The hypersensitivity results confirm that thyroid hormone stimulation of malic enzyme synthesis occurs in part at the level of transcription, and localization of malic enzyme gene expression suggests this stimulation is accompanied by recruitment of hepatocytes. Hepatocytes may be heterogeneous in their ability to respond to thyroid hormone.

A pH Control System Based on Malate Decarboxylation for the Cultivation of Lactic Acid Bacteria

Most species of lactic acid bacteria decarboxylate l-malate to lactate and CO(2) if an energy source such as glucose is present. A proton is taken up in the reaction, which prevents pH decreases in the growth medium caused by lactic acid production from glucose fermentation. MRS broth (pH 7.0) (Difco Laboratories) containing 10 mM glucose and various concentrations of l-malate (0, 25, 50, 75, and 100 mM) was used to cultivate Lactobacillus plantarum. After 72 h at 37 degrees C, all malate was decarboxylated and all glucose was fermented, with resultant final pH values of 4.5, 6.3, 6.9, 7.3, and 7.5, respectively. When d-malate (which cannot be decarboxylated) was substituted for l-malate, the final pH values were 4.5, 5.2, 5.6, 5.8, and 5.9. By varying the ratios of glucose to l-malate in the growth medium, it was possible to obtain pH values which were lower, the same, or higher than the initial pH values. In contrast, buffers such as phosphate only retard decreases in pH. l-Malate, when compared with K(2)PO(4) on an equal molar basis, provided greater resistance to decreases in pH. Higher specific growth rates were observed for L. plantarum and Leuconostoc mesenteroides when l-malate rather than K(2)PO(4) was incorporated into the growth medium.

Isotope effect studies of chicken liver NADP malic enzyme: role of the metal ion and viscosity dependence.

The role of the metal ion in the oxidative decarboxylation of malate by chicken liver NADP malic enzyme and details of the reaction mechanism have been investigated by 13C isotope effects. With saturating NADP and the indicated metal ion at a total concentration 10-fold higher than its Km, the following primary 13C kinetic isotope effects at C4 of malate [13(V/Kmal)] were observed at pH 8.0: Mg2+, 1.0336; Mn2+, 1.0365; Cd2+, 1.0366; Zn2+, 1.0337; Co2+, 1.0283; Ni2+, 1.025. Knowing the partitioning of the intermediate oxalacetate between decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation. For Mg2+ as activator, this was 1.049 with NADP and 1.046 with 3-acetylpyridine adenine dinucleotide phosphate, although the intrinsic primary deuterium isotope effects on dehydrogenation were 5.6 and 4.2, and the partition ratios of the oxalacetate intermediate for decarboxylation as opposed to hydride transfer were 0.11 and 3.96 (the result of the different redox potentials of NADP and the acetylpyridine analogue). The close agreement of the intrinsic 13C isotope effects with each other and with the 13C isotope effect for the Mg2+-catalyzed nonenzymatic decarboxylation of oxalacetate of 1.0489 [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582] indicates a similarity of transition states for these reactions. It was not possible to calculate reasonable intrinsic carbon isotope effects with the other metal ions by use of the partitioning ratio of oxalacetate because of decarboxylation by another mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of malolactic fermentation in lactic acid bacteria

Although decarboxylation of malate to lactate by malolactic enzyme does not liberate biologically available energy ( e.g., ATP, NADH), the growth rate of many malolactic bacteria is greatly enhanced by malolactic fermentation. The deacidification of the medium due to malate dissipation cannot fully account for this situation. The chemiosmotic theory postulates that another form of energy could generated by translocation of protons through the membrane coupled to end-product efflux. Konings et al. showed that this theory is indeed applicable to lactate efflux in Streptococcus cremoris at pH 7.0 A similar mechanism could account for the observed increased activity in malolactic bacteria. The study in wild type and mutant strains of Streptococcus lactis unable to carry out malolactic fermentation led us to the following conclusions: (1) under glucose non-limiting conditions, malolactic fermentation helps to maintain pH of the medium at a certain level; (2) during glucose limited growth, malolactic fermentation could be coupled with an energetic process independent from that mentioned above.

C3and CAM Photosynthetic Characteristics of the Submerged Aquatic MacrophyteLittorella uniflora: Regulation of Leaf Internal CO2Supply in Response to Variation in Rooting Substrate Inorganic Carbon Concentration

The relationships between CO2 concentrating mechanisms, photosynthetic efficiency and inorganic carbon supply have been investigated for the aquatic macrophyte Littorella uniflora. Plants were obtained from Esthwaite Water or a local reservoir, with the latter plants transplanted into a range of sediment types to alter CO2 supply around the roots. Free CO2 in sediment-interstitial-water ranged from 1–01 mol m−3 (Esthwaite), 0.79 mol m−3 (peat), 0.32 mol m−3 (silt) and 0–17 mol m−3 (sand), with plants maintained under PAR of 40 μmol m−2 s−1. A comparison of gross morphology of plants maintained under these conditions showed that the peat-grown plants with high sediment CO2 had larger leaf fresh weight (0–69 g) and total surface area (223 cm2 g−1 fr. wt. including lacunal surface area) than the sand-grown plants (0.21 g and 196 cm2 g−1 fr. wt. respectively). Root fresh weights were similar for all treatments. In contrast, leaf internal CO2 concentration [CO2], was highest in the sand-grown plants (2–69 mol m−3, corresponding to ∼ 6.5% CO2 in air) and lowest in the Esthwaite plants (1–08 mol m−3). Expression of CAM in transplants was also greatest in the low CO2 regime, with ΔH+ (measured as dawn-dusk titratable acidity) of 50μmol g− fr. wt., similar to Esthwaite plants in natural sediment. Assuming typical CAM stoichiometry, decarboxylation of malate could account largely for the measured [CO2]1 and would make a major contribution to daytime CO2 fixation in vivo. A range of leaf sections (0–2, 1–0, 5–0 and 17–0 mm) was used to evaluate diffusion limitation and to select a suitable size for comparative studies of photosynthetic O2 evolution. The longer leaf sections (17.0 mm), which were sealed and included the leaf tip, were diffusion-limited with a linear response to incremental addition of CO2 and 1–0 mol m−3 exogenous CO2 was required to saturate photosynthesis. Shorter leaf sections were less diffusion-limited, with the greatest photosynthetic capacity (36 μmol O2 g−1 fr. wt. h−1) obtained from the 1.0 mm size and were not infiltrated by the incubating medium. Comparative studies with 1.0 mm sections from plants grown in the different sediment types revealed that the photosynthetic capacity of the sand-grown plants was greatest (45 μmol O2 g−1 fr. wt. h−1) with a K0.5 of 80 mmol m−3. In terms of light response, saturation of photosynthesis in tissue slices occurred at 850–1000 μmol m−2 s−1 although light compensation points (6–11 μmol m−2s−1) and chlorophyll a: b ratios (1.3) were low. While CO2 and PAR responses were obtained using varying numbers of sections with a constant fresh weight, the relationships between photosynthetic capacity and CO2 supply or PAR were maintained when the data were expressed on a chlorophyll basis. It is concluded that under low PAR, CO2 concentrating mechanisms interact in intact plants to maintain saturating CO2 levels within leaf lacunae, although the responses of the various components of CO2 supply to PAR require further investigation.