Comparison of glycolytic and associated enzyme activities in the claw and tail muscles of the lobster with those of the mantle of Mytilus edulis

In the mussel Mytilus edulis, the activities of phosphoenolpyruvate car?ykinase (PEP-CK) and of malate dehydrogenase (MDH) are greater than those of pyruvate kinase (PK) and lactate dehydrogenase (LDH). The activities of PEP-CK are very low in the lobster (H. vulgaris.) and the ratio of MDH/LDH activities are 0·041 and 1·83 in the tail and the claw muscles respectively. Intracellular L-lactate and L-alanine concentrations suggest a different carbohydrate utilization in the tail and the claw muscle of the lobster. Consistent with this finding is the fact that L-lactate concentration is higher in the tail muscle than in the claw muscle; the opposite is true for L-alanine concentrations.     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S_0.5 value 10³-fold and increased the V_max value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His¹⁷⁹ of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V_max 4-fold but reduced pyruvate S_0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V_max, but 10²-reduced pyruvate S_0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.     

Glycolytic activity of the claw and tail muscle homogenates of the lobster, Homarus vulgaris

The fate of D-glucose-6-phosphate and phosphoenolpyruvate in homogenates of tail and claw muscles of the lobster (H vulgaris) has been studied. With both substrates oxygen utilization is higher for the claw than for the tail muscle. Succinate is not an end product of anaerobic D-glucose-6-phosphate-U- ¹⁴C degradation in either muscle but L-lactate is the major product of such catabolism in tail muscle whereas both L-lactate and L-alanine are produced in claw muscle. Dihydroxyacetone phosphate production was observed both tissues: this can be related to the known high lipid content of these tissues.     

L(+)-lactate dehydrogenase of Clostridium acetobutylicum is activated by fructose-1,6-bisphosphate

Cells of Clostridium acetobutylicum contained an NADH-dependent L(+)-lactate dehydrogenase which was activated specifically by fructose-1,6-bisphosphate (F-1,6-P₂), with calcium or magnesium ions as positive effectors. During the purification steps the enzyme was very unstable. The purified enzyme existed in a tetrameric structure (apparent M_r of about 159 kDa) and had its pH optimum at pH 5.8. Little activity was left at pH values below 5.0. The enzyme was unidirectional, catalysing only the reduction of pyruvate. The half maximal activation of the reaction velocity with F-1,6-P₂ depended on the pyruvate concentration.     

The respiratory system of the marine bacterium Beneckea natriegens. II. Terminal branching of respiration to oxygen and resistance to inhibition by cyanide

1. Cell-free extracts of the marine bacterium Beneckea natriegens, derived by sonication, were separated into particulate and supernatant fractions by centrifugation at 150 000 × g.2. NADH, succinate, d(?)- and l(+)-lactate oxidase and dehydrogenase activities were located in the particles, with 2- to 3-fold increases in specific activity over the cell free extract. The d(?)- and l(+)-lactate dehydrogenases were NAD⁺ and NADP⁺ independent. Ascorbate-N,N,N′,N′-tetramethylphenylenediamine (TMPD) oxidase was also present in the particulate fraction; it was 7–12 times more active than the physiological substrate oxidases.3. Ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide. Succinate, NADH, d(?)-lactate and l(+)-lactate oxidases were inhibited in a biphasic manner, with 10 μM cyanide causing only 10–50 % inhibition; further inhibition required more than 0.5 mM cyanide, and 10 mM cyanide caused over 90 % inhibition. Low sulphide (5 μM) and azide (2 mM) concentrations also totally inhibited ascorbate-TMPD oxidase, but only partially inhibited the other oxidases. High concentrations of sulphide but not azide caused a second phase inhibition of NADH, succinate, d(?)-lactate and l(+)-lactate oxidases.4. Low oxidase activities of the physiological substrates, obtained by using non-saturating substrate concentrations, were more inhibited by 10 μM cyanide and 2 mM azide than high oxidase rates, yet ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide over a wide range of rates of oxidation.5. These results indicate terminal branching of the respiratory system. Ascorbate-TMPD is oxidised by one pathway only, whilst NADH, succinate, d(?)-lactate and l(+)-lactate are oxidised via both pathways. Respiration of the latter substrates occurs preferentially by the pathway associated with ascorbate-TMPD oxidase and which is sensitive to low concentrations of cyanide, azide and sulphide.6. The apparent K_m for O₂ for each of the two pathways was detected using ascorbate-TMPD and NADH or succinate plus 10 μM cyanide respectively. The former pathway had an apparent K_m of 8–17 (average 10.6) μM and the latter 2.2–4.0 (average 3.0) μM O₂.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH₄⁺ in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH₄⁺ to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH₄⁺ assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH₄⁺ assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH₄⁺ assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH₄⁺ assimilation.     

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD⁺-dependent l-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator d-fructose-1,6-bisphosphate (Fru-1,6-P₂). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with V _max for the reduction of pyruvate being approximately tenfold that of the value for l-lactate oxidation at pH 7.5. No deviation from Michaelis–Menten kinetics was observed in the presence of Fru-1,6-P₂, while a sigmoidal response (indicative of positive cooperativity) was seen towards l-lactate without Fru-1,6-P₂. Strikingly, when introduced into an lldD ⁻ strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on l-lactate as the sole carbon source.     

Urea-requiring lactate dehydrogenases of marine elasmobranch fishes

Summary The kinetic properties — apparentK _m of pyruvate, pyruvate inhibition pattern, and maximal velocity — of M₄ (skeletal muscle) lactate dehydrogenases of marine elasmobranch fishes resemble those of the homologous lactate dehydrogenases of non-elasmobranchs only when physiological concentrations of urea (approximately 400 mM) are present in the assay medium. Urea increases the apparentK _m of pyruvate to values typical of other vertebrates (Fig. 2), and reduces pyruvate inhibition to levels seen with other M₄-lactate dehydrogenases (Fig. 3). Urea reduces the activation enthalpy of the reaction, and increasesV _max at physiological temperatures (Fig. 4).The M₄-lactate dehydrogenase of the freshwater elasmobranch,Potamotrygon sp., resembles a teleost lactate dehydrogenase, i.e., although it is sensitive to urea, it does not require the presence of urea for the establishment of optimal kinetic properties.     

Influence of copper and zinc ions on toxicity of sodium N-methyldithiocarbamate to Fusarium oxysporum f. sp. lycopersici

Summary It was found that dl-lactate, acetate, pyruvate, succinate, fumarate, malate, oxalacetate and L-aspartate stimulated considerably the production of the antitumor L-asparaginase EC-2 by a colicinogenic strain of E. coli in Tryptone medium. While in the absence of these stimulative substances enzyme production was about 0.01 IU/mg dry weight, it reached 0.57–1.18 IU/mg in the presence of the substances named above. Maximum effects were obtained at the concentrations 0.025–0.1 M; at higher concentrations enzyme production decreased. L-asparagine raised the enzyme production from 0.05 IU/mg to 0.70 IU/mg; in order to obtain this effect, however, it was necessary to lower the oxygen absorption rate. Citrate and -ketoglutarate had no stimulatory effect; the effect of L-glutamate was negligible. In the cases of dl-lactate, pyruvate and L-aspartate the dependence of stimulatory effects on the time of addition of the stimulants was studied. It was found that in the case of pyruvate and especially of dl-lactate enzyme production increased rapidly between the 4 and 6 h of cultivation, i.e. at the end of the exponential growth phase; it did not matter whether the stimulant had been added at zero, 2 or 4 h of cultivation. In the case of L-aspartate the situation was principally similar, though the enzyme production started later and increased more slowly.     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^? pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono-γ-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

Modeling of the pyruvate production with<Emphasis Type="Italic"> Escherichia coli</Emphasis> in a fed-batch bioreactor

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q_VG=10 mL h^–1. Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q_VG=20 and 30 mL h^–1, respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking–Piret/Levenspiel term).List of symbols c_A acetate concentration (g L^–1) - c_A,0 acetate concentration in the feed (g L^–1) - c_G glucose concentration (g L^–1) - c_G,0 glucose concentration in the feed (g L^–1) - c_P pyruvate concentration (g L^–1) - c_P,max critical pyruvate concentration above which reaction cannot proceed (g L^–1) - c_X biomass concentration (g L^–1) - K_I inhibition constant for pyruvate production (g L^–1) - K_I^A inhibition constant for biomass growth on acetate (g L^–1) - K_P saturation constant for pyruvate production (g L^–1) - K_P inhibition constant of Jerusalimsky (g L^–1) - K_S^A Monod growth constant for acetate (g L^–1) - K_S^G Monod growth constant for glucose (g L^–1) - m_A maintenance coefficient for growth on acetate (g g^–1 h^–1) - m_G maintenance coefficient for growth on glucose (g g^–1 h^–1) - n constant of extended Monod kinetics (Levenspiel) (–) - q_V volumetric flow rate (L h^–1) - q_VA volumetric flow rate of acetate (L h^–1) - q_VG volumetric flow rate of glucose (L h^–1) - r_A specific rate of acetate consumption (g g^–1 h^–1) - r_G specific rate of glucose consumption (g g^–1 h^–1) - r_P specific rate of pyruvate production (g g^–1 h^–1) - r_P,max maximum specific rate of pyruvate production (g g^–1 h^–1) - t time (h) - V reaction (broth) volume (L) - Y_P/G yield coefficient pyruvate from glucose (g g^–1) - Y_X/A yield coefficient biomass from acetate (g g^–1) - Y_X/A,max maximum yield coefficient biomass from acetate (g g^–1) - Y_X/G yield coefficient biomass from glucose (g g^–1) - Y_X/G,max maximum yield coefficient biomass from glucose (g g^–1) - growth associated product formation coefficient (g g^–1) - non-growth associated product formation coefficient (g g^–1 h^–1) - specific growth rate (h^–1) - _max maximum specific growth rate (h^–1)     

Corynebacterium glutamicum tailored for efficient isobutanol production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y_P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y_P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y_P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H⁺ to NADPH+H⁺. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y_P/S of about 0.48 mol per mol of glucose in the production phase.     

The oxidation of d- and l-glycerate by rat liver

1. The interconversion of hydroxypyruvate and l-glycerate in the presence of NAD and rat-liver l-lactate dehydrogenase has been demonstrated. Michaelis constants for these substrates together with an equilibrium constant have been determined and compared with those for pyruvate and l-lactate. 2. The presence of d-glycerate dehydrogenase in rat liver has been confirmed and the enzyme has been purified 16–20-fold from the supernatant fraction of a homogenate, when it is free of l-lactate dehydrogenase, with a 23–29% recovery. The enzyme catalyses the interconversion of hydroxypyruvate and d-glycerate in the presence of either NAD or NADP with almost equal efficiency. d-Glycerate dehydrogenase also catalyses the reduction of glyoxylate, but is distinct from l-lactate dehydrogenase in that it fails to act on pyruvate, d-lactate or l-lactate. The enzyme is strongly dependent on free thiol groups, as shown by inhibition with p-chloromercuribenzoate, and in the presence of sodium chloride the reduction of hydroxypyruvate is activated. Michaelis constants for these substrates of d-glycerate dehydrogenase and an equilibrium constant for the NAD-catalysed reaction have been calculated. 3. An explanation for the lowered V_max. with d-glycerate as compared with dl-glycerate for the rabbit-kidney d-α-hydroxy acid dehydrogenase has been proposed.     

Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway

In previous research, a thiamine-auxotrophic yeast for alpha-ketoglutaric acid (KGA) overproduction was screened in our laboratory and named Yarrowia lipolytica WSH-Z06 (CCTCC no. M207143). However, the high concentration of by-products (mainly pyruvate) limited its application on an industrial scale. To enhance KGA production and reduce pyruvate (PA) accumulation, the pyruvate carboxylation pathway was regulated. By overexpressing the pyruvate carboxylase genes ScPYC1 from Saccharomyces cerevisiae and RoPYC2 from Rhizopus oryzae in Y. lipolytica WSH-Z06, the yields of KGA in Y. lipolytica-ScPYC1 and Y. lipolytica-RoPYC2 increased by 24.5 and 35.3?%, and the yields of PA decreased by 51.9 and 69.8?% in shake flasks, respectively. These changes in the expression levels and activities of key intracellular enzymes showed that enhancing the pyruvate carboxylation pathway had successfully redistributed the carbon flux from PA to KGA. Finally, by controlling the pH in a 3-L fermenter, the maximum concentration of KGA in Y. lipolytica-RoPYC2 reached 62.5?g?L^?1 with an evident decrease in PA yield from 35.2 to 13.5?g?L^?1.     

<Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> as a novel biocatalyst for pyruvate production from<Emphasis Type="SmallCaps"> DL</Emphasis>-lactate

Pseudomonas stutzeri SDM oxidized dl-lactic acid (25.5 g l^-1) into pyruvic acid (22.6 g l^-1) over 24 h. Both NAD⁺-independent d-lactate dehydrogenase and NAD⁺-independent l-lactate dehydrogenase were found for the first time in the bioconversion of lactate to pyruvate based on the enzyme activity assay and proteomic analysis. Jianrong Hao and Cuiqing Ma contributed equally to this work     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : II. Inhibition of Apparent Photosynthesis by a Phosphoenolpyruvate Carboxylase Inhibitor

Hybrids have been made between species of Flaveria exhibiting varying levels of C₄ photosynthesis. The degree of C₄ photosynthesis expressed in four interspecific hybrids (Flaveria trinervia [C₄] × F. linearis [C₃-C₄], F. brownii [C₄-like] × F. linearis, and two three-species hybrids from F. trinervia × [F. brownii × F. linearis]) was estimated by inhibiting phosphoenolpyruvate carboxylase in vivo with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP). The inhibitor was fed to detached leaves at a concentration of 4 mm, and apparent photosynthesis was measured at atmospheric levels of CO₂ and at 20 and 210 mL L⁻¹ of O₂. Photosynthesis at 210 mL L⁻¹ of O₂ was inhibited 32% by DCDP in F. linearis, by 60% in F. brownii, and by 87% in F. trinervia. Inhibition in the hybrids ranged from 38 to 52%. The inhibition of photosynthesis by 210 mL L⁻¹ of O₂ was increased when DCDP was used, except in the C₄ species, F. trinervia, in which photosynthesis was insensitive to O₂. Except for F. trinervia, control plants with less O₂ sensitivity (more C₄-like) exhibited a progressively greater change in O₂ inhibition of photosynthesis when treated with DCDP. This increased O₂ inhibition probably resulted from decreased CO₂ concentrations in bundle sheath cells due to inhibition of phosphoenolpyruvate carboxylase. The inhibition of photosynthesis by DCDP is concluded to underestimate the degree of C₄ photosynthesis in the interspecific hybrids because increased direct assimilation of atmospheric CO₂ by ribulose bisphosphate carboxylase may compensate for inhibition of phosphoenolpyruvate carboxylase.     

Involvement of na in active uptake of pyruvate in mesophyll chloroplasts of some c(4) plants : na/pyruvate cotransport

An artificial Na⁺ gradient across the envelope (Na⁺ jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C₄ plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219:347). In the present study, ²²Na⁺ and pyruvate uptake were examined in mesophyll chloroplasts of several species of C₄ plants. Enhancement of pyruvate uptake by a Na⁺ jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in turn enhanced Na⁺ uptake in the dark when added together with Na⁺. When flux of endogenous Na⁺ was measured in these mesophyll chloroplasts preincubated with ²²Na⁺, pyruvate addition induced Na⁺ influx, and the extent of the pyruvate-induced Na⁺ influx positively correlated with that of pyruvate uptake. A Na⁺/H⁺ exchange ionophore, monensin, nullified all the above mutual effects of Na⁺ and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na⁺ uptake and increased equilibrium level of chloroplast ²²Na⁺. Measurements of initial uptake rates of pyruvate and Na⁺ gave a stoichiometry close to 1:1. These results point to Na⁺/pyruvate cotransport into mesophyll chloroplasts of some C₄ plants.