Oxygen evolution by a reconstituted spinach chloroplast system in the presence ofl-glutamine and 2-oxoglutarate

Intact chloroplasts prepared from summer-grown spinach plants supported (aspartate plus 2-oxoglutarate)-dependent O₂ evolution but not (glutamine plus 2-oxoglutarate)-dependent O₂ evolution. The former activity, which was sensitive to amino oxyacetate, was attributed to transaminase activity and reduction of the resulting oxalo-acetate to malate using H₂O as eventual electron donor. A reconstituted chloroplast system which included chloroplast stroma, thylakoid membranes, ferredoxin and NADP(H) supported O₂ evolution in the presence ofl-glutamine and 2-oxoglutarate at rates of 15–22 μmol mg^-1 chlorophyll h^-1 although lower rates were obtained with material from winter-grown plants. Activity was not observed in the absence of ferredoxin and omission of NADP(H) decreased activity by 40%. The reaction was associated with the production of 0.49 mol O₂ mol^-1 2-oxoglutarate consumed and up to 0.46 mol O₂ mol^-1 glutamine supplied. The reaction, which was inhibited by azaserine but not by methionine sulphoximine or amino oxyacetate, was attributed to light-coupled glutamate synthase (EC 1.4.1.13) with H₂O serving as eventual electron donor. Activity was not affected significantly byl-malate. The reconstituted system also supported O₂ evolution in the presence of nitrite, oxaloacetate, (aspartate plus 2-oxoglutarate) and oxidised glutathione.     

Light-dependent reduction of pyruvate by pea chloroplasts in the presence of glutamate dehydrogenase and C5-dicarboxylic acids

Illuminated pea chloroplasts supported (glutamine plus α-oxoglutarate (α-OG)) and (NH₃ plus α-OG)-dependent O₂ evolution. The properties of these reactions were consistent with light-coupled glutamate synthase and glutamine synthetase activities. In the presence of a glutamate-oxidizing system (component C) comprised of NAD-specific glutamate dehydrogenase (NAD-GDH), lactate dehydrogenase (LDH), 4 mM pyruvate and 0.2 mM NAD, illuminated chloroplasts supported O₂ evolution in the presence of glutamine. The reaction did not proceed in the absence of any one of the constituents of component C and the properties of O₂ evolution were consistent with light-coupled glutamate synthase activity. In the presence of component C, chloroplasts also catalysed O₂ evolution in the presence of catalytic concentrations of glutamate. Studies of O₂ evolution and metabolism of [¹⁴C]-glutamate in the presence of the inhibitors methionine sulphoximine (MSO) and azaserine suggest that O₂ evolution was dependent on the synthesis of glutamine from the products of glutamate oxidation. This was supported by polarographic studies using α-OG and NH₃ instead of glutamate.The results are consistent with a C₅-dicarboxylic acid shuttlemechanism for the export of reducing equivalents from illuminated chloroplasts (glutamate) and recycling of the oxidation products (α-OG and NH₃).     

Characterization of dicarboxylate stimulation of ammonia, glutamine, and 2-oxoglutarate-dependent o(2) evolution in isolated pea chloroplasts

Intact isolated chloroplasts from pea (Pisum sativum) leaves carried out light-dependent (NH₃, 2-oxoglutarate) and (glutamine, 2-oxoglutarate)-dependent O₂ evolution at rates of 3.3 ± 0.7 (n = 7) and 6.0 ± 0.4 (n = 5) micromoles per milligram chlorophyll per hour, respectively. Malate stimulated the rate of (NH₃, 2-oxoglutarate)-dependent O₂ evolution 2.1 ± 0.5 (n = 7)-fold in the absence of glutamine, and 3.3 ± 0.4 (n = 11)-fold in the presence of glutamine. Malate also stimulated (glutamine, 2-oxoglutarate)-dependent O₂ evolution in the presence of high concentrations of glutamine. The affinity (K_1/2) of (NH₃, glutamine, 2-oxoglutarate)-dependent O₂ evolution for 2-oxoglutarate was estimated at 200 to 250 micromolar in the absence of malate and 50 to 80 micromolar when malate (0.5 millimolar) was present. In contrast to malate and various other dicarboxylates, aspartate, glutarate, and glutamate did not stimulate (NH₃, glutamine, 2-oxoglutarate)-dependent O₂ evolution in isolated pea chloroplasts. Using both in vitro assays and reconstituted chloroplast systems, malate was shown to have no effect on the activities of either glutamine synthetase or glutamate synthase.

The concentration of malate required for maximal stimulation of O₂ evolution was dependent on the concentration of 2-oxoglutarate present. However, the small extent of the competition between malate and 2-oxoglutarate for uptake was not consistent with that predicted by the current `single carrier' model proposed for the uptake of dicarboxylates into chloroplasts.

     

Effect of inhibitors on ammonia-, 2-oxoglutarate-, and oxaloacetate-dependent o(2) evolution in illuminated chloroplasts

The evolution of O₂ in spinach chloroplasts in the presence of oxaloacetate (OAA) was inhibited by a wide range of dicarboxylates. In contrast, (ammonia, 2-oxoglutarate)-dependent O₂ evolution was stimulated by malate, succinate, fumarate, glutarate, maleiate, and l-tartrate although OAA has little effect. This increase in O₂ evolution was accompanied by a similar increase in ¹⁴C incorporation from [5-¹⁴C]oxoglutarate into amino acids which was sensitive to azaserine inhibition. Glutamate and aspartate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution, but this inhibition was relieved by the addition of succinate, malate, or fumarate. OAA-dependent O₂ evolution also was inhibited by glutamate and aspartate, but succinate, malate, or fumarate had little effect on this inhibition. Phthalonate and n-butyl malonate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution competitively with respect to 2-oxoglutarate and uncompetitively with respect to malate. Both these inhibitors inhibited OAA-dependent O₂ evolution competitively. This evidence suggests that different mechanisms might be involved in the transport of OAA, 2-oxoglutarate, and malate into the chloroplasts.     

A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH₄Cl preincubation in the light. Treatment with NH₄Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH₄Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH₄Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH₃, 2-OG)-dependent O₂ evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH₃ assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.     

Correlation between photosynthesis and the transthylakoid proton gradient

In isolated intact chloroplasts, maximal rates of photosynthetic O₂ evolution (in saturating HCO^?₃) are associated with a critical transthylakoid proton gradient as a result of the stoichiometric consumption of 2 mol NADPH and 3 mol ATP/mol CO₂ fixed. Studies with the fluorescent probe 9-aminoacridine reveal that in the illuminated steady state the critical ΔpH is 3.9.CO₂-dependent O₂ evolution is inhibited by increases of 0.1–0.2 in ΔpH that occur when catalase is omitted from the medium, NO^?₂ is included as an electron acceptor, or when chloroplasts are illuminated under low partial pressures of O₂. Low concentrations of antimycin (0.33 μM) or NH₄Cl (0.33 mM) decrease ΔpH and relieve this inhibition of electron flow. The energy transfer inhibitor quercetin lowers the high ATP/ADP ratio associated with these conditions, but does not lower ΔpH or relieve the inhibition.A decrease of ΔpH below 3.9 by weaker illumination, millimolar levels of NH₄Cl or micromolar levels of antimycin, results in lower rates of photosynthesis owing to limitation by the phosphorylation rate.These findings show that in absence of rate limitation by the carbon cycle, the extent of thylakoid energization is related to the ratio of ATP to NADPH production and in turn, the rate of CO₂ assimilation.     

N-ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light

The direct incorporation of ¹⁵NH₄Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during ¹⁵NH₄ assimilation in the presence of malate. In 4 minutes the ¹⁵N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of ¹⁵NH₄Cl and Na¹⁵NO₂ greatly increased the ¹⁵N-label into glutamine but did not qualitatively change the order of the incorporation of ¹⁵N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in [¹⁴C]2-oxoglutarate transport into the chloroplast as well as an increase in the export of [¹⁴C]glutamate out of the chloroplast. Little [¹⁴C]glutamine was detected in the medium of the chloroplast preparations. The stimulation of ¹⁵N-incorporation and [¹⁴C]glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2-oxoglutarate translocator) observed in the presence of exogenous malate.     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

Inhibition of chlorophyll biosynthesis by lead in greeningPisum sativum leaf segments

Supply of 0.01 to 1.0 mM lead acetate to greening pea(Pisum sativum L.) leaf segments either in the absence or in the presence of inorganic nitrogen lowered total chlorophyll (Chl) content. During a time course study, there was not any appreciable effect of Pb²⁺ upto 4 h but thereafter Pb inhibited Chl synthesis. While supply of succinate, cysteine dithiothreitol, 5,5-dithio-bis-2-nitrobenzoic acid and NH₄C1 had no protective action against Pb²⁺ toxicity, and glycine, glutamate 2-oxoglutarate, MgCl₂, KH₂PO₄, CaCl₂, KC1 protected only partially, reduced glutathione (GSH) could completely overcome the inhibition of Chl biosynthesis by the metal. It is suggested that Pb²⁺ interferes with Chl biosynthesis through GSH availability     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

A method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of ¹⁴CO₂ fixation both in the light (45 μmol of CO₂ fixed mg^?1 Chl h^?1) and in the dark (20 μmol of CO₂ fixed mg^?1 Chl h^?1). The protoplasts also showed O₂ evolution (40 μmol of O₂ evolved mg^?1 Chl h^?1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O₂ evolution. Analyses of early photosynthetic products in the light showed the formation of both C₃ and C₄ acids. Aspartate was found to be a predominant photosynthate.     

Glutamate synthase, but not GABA shunt enzymes, contributes to nitrogen metabolism of the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

Glutamate synthase (E.C. 1.4.1.14) (GOGAT) activity was not detectable in L3 Haemonchus contortus, but was present in L3 Teladorsagia circumcincta and adult worms of both species. GOGAT activity was inhibited by 80% by azaserine. Activity (nmol min⁻¹ mg⁻¹ protein) was 33–59 in adult H. contortus, 51–91 in adult T. circumcincta and 24–41 in L3 T. circumcincta, probably depending on exposure to ammonia, as incubation with 1 mM NH₄Cl doubled GOGAT activity. The pH optimum was 7.5 in both species. Either NAD or NADP acted as co-factor. The mean apparent K_m for 2-oxoglutarate was 0.7 (0.5–0.9) mM and for glutamine was 1.0 (0.5–1.7) mM for different homogenates. There was no detectable activity in whole parasite homogenates of glutamate decarboxylase (E.C. 4.1.1.15) or succinic semialdehyde dehydrogenase (E.C. 1.2.1.24), the first and third enzymes of the GABA shunt, respectively, suggesting that the GABA shunt is not important in general metabolism in these species.     

Characterization of a light-dependent glutamate synthase activity in Chlamydomonas reinhardtii

Photosynthetically active vesicles prepared from Chlamydomonas reinhardtii retained a light-dependent glutamate synthase activity which was highly specific for 2-oxoglutarate (K_m=2.1 mM) and L-glutamine (K_m=0.9 mM) as amido group acceptor and donor respectively. This activity was inhibited by azaserine, p-hydroxymercuribenzoate and 3-(p-chlorophenyl)-1,1-dimethyl urea.Light-dependent synthesis of glutamate was also obtained by coupling Chlamydomonas photosynthetic particles to purified ferredoxin-glutamate synthase, using ascorbate and 2,6-dichlorophenol-indophenol as electron donor. This system was also specific for 2-oxoglutarate (K_m=1 mM) and L-glutamine (K_m=0.8 mM) as substrates, and was stimulated by dithioerythritol. Azaserine and p-hydroxymercuribenzoate, but not 3-(p-chlorophenyl)-1,1-dimethyl urea, inhibited the reconstituted activity; high concentrations of 2-oxoglutarate were inhibitory.Abbreviations A Absorbance - CCP p-Trichlorometoxi-carbonylcyanide-phenylhydrazone - Chl Chlorophyll - CMU 3-(p-Chlorophenyl)-1,1-dimethyl urea - DPIP 2,6-Dichlorophenol-indophenol - DTE Dithioerythritol - MSX L-Methionine, D-L, sulfoximine - MV Methyl viologen     

Photosynthesis and phosphorylation of light-harvesting chlorophyll a/b—protein in intact chloroplasts: Effects of uncouplers

Protein phosphorylation in isolated, intact pea chloroplasts was measured during the onset of CO₂-dependent O₂ evolution. Total incorporation of ³²P (from ³²P_i) into the light-harvesting chlorophyll a/b—protein was found to be less sensitive than O₂ evolution to inhibition by the uncouplers FCCP and NH₄C1 It is concluded that changes in the rate of ATP synthesis cannot affect protein phosphorylation without also affecting the rate of CO₂-fixation in this system. The ATP/ADP ratio is therefore unlikely to regulate photosynthetic protein phosphorylation under normal physiology conditions.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

Growth and fermentation responses of Selenomonas ruminantium to limiting and non-limiting concentrations of ammonium chloride

 The objective of this study was to assess fermentation product, growth rate and growth yield responses of Selenomonas ruminantium HD₄ to limiting and non-limiting ammonia concentrations. The ammonia half-inhibition constant for S. ruminantium in batch culture was 296 mM. Cells were grown in continuous culture with a defined ascorbate-reduced basal medium containing either 0.5, 5, 25, 50, 100 or 200 mM NH₄Cl and dilution rates were 0.07, 0.14, 0.24 or 0.40 h^-1. Ammonia was the growth-limiting nutrient when 0.5 mM NH₄Cl was provided and the half-saturation constant was 72 μM. Specific rates of glucose utilization and fermentation acid carbon formation were highest for 0.5 mM NH₄Cl. Lactate production (moles per mole of glucose disappearing) increased at the fastest dilution rate (0.40 h^-1) for 5.0 mM NH₄Cl while acetate and propionate decreased when compared to slower dilutions (0.07 and 0.14 h^-1). Lactate production remained low while acetate and propionate remained high for all dilution rates when NH₄Cl concentrations were 25 mM or greater. Yield (Y _Glc and Y _ATP) were nearly doubled when NH₄Cl was increased from 0.5 mM (25.1 g cells/mol glucose used and 13.9 g cells/mol ATP produced respectively) to the higher concentrations. Y _Glc was highest at 25 mM and 50 mM NH₄Cl (48.2 cells/mol and 43.1 cells/mol respectively) as was Y _ATP (23.2 cells/mol and 20.8 cells/mol respectively). Y _NH3 was highest at the lowest NH₄Cl concentration. The maximal fermentation product formation rate occurred at a growth-limiting ammonia concentration, while maximal glucose and ATP bacterial yields occurred at non-growth-limiting ammonia concentrations. Given the growth response of this ruminal bacterium, it is possible that maximization of ruminal bacterial yield may necessitate sacrificing the substrate degradation rate and vice versa. Received: 5 December 1995/Received revision: 2 April 1996/Accepted: 22 April 1996     

Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

Light-dependent reduction of hydrogen peroxide by intact spinach chloroplasts

Intact spinach chloroplasts, washed four times in buffered sorbitol to decrease catalase contamination, supported O₂ evolution in the dark at very low rates (less than 2 μmol/mg Chl per h) in the presence of low concentrations of H₂O₂ (0.25 mM); H₂O₂ was not significantly metabolished under these conditions. In the light, washed chloroplasts supported H₂O₂-dependent O₂ evolution at rates of 28–46 μmol/mg Chl per h in the presence of 0.1–0.25 mM H₂O₂; the concentration of H₂O₂ supporting 0.5V_max was estimated to be 25 μM. O₂ evolution in the light was associated with H₂O₂ consumption and ceased after the production of 0.45 mol per mol H₂O₂ consumed. Both O₂ evolution and H₂O₂ consumption were abolished by 5 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Washed intact chloroplasts contained endogenous pools of GSH and ascorbate estimated at 10 and 33 mM, respectively. H₂O₂-dependent O₂ evolution in the light was associated with a decrease in these levels which increased as O₂ evolution gradually ceased. The results are consistent with the hypothesis that H₂O serves as eventual electron donor for the reduction of H₂O₂ in illuminated chloroplasts and that GSH/GSSG and ascorbate/dehydroascorbate serve as intermediate electron carriers. Preincubation of chloroplasts in the dark with 0.1 mM H₂O₂ abolished O₂ evolution in the light.