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1.
Neither Dunaliella cells grown with 5% CO 2 nor their isolated chloroplasts had a CO 2 concentrating mechanism. These cells primarily utilized CO 2 from the medium because the K(0.5) (HCO 3−) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K(0.5) CO 2 was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO 2 concentrating mechanism was present that decreased the K0.5 (CO 2) to about 0.5 micromolar and K0.5 (HCO 3−) to 11 micromolar at pH 8. These K0.5 values suggest that air-adapted cells preferentially concentrated CO 2 but could also use HCO 3− from the medium. Chloroplasts isolated from air-adapted cells had a K(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO 2. Chloroplasts from air-adapted cells, but not CO 2-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO 2 concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO 2 concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO 2 concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport. 相似文献
2.
A particulate preparation (MgP) capable of photosynthetic CO 2 assimilation without the addition of stromal protein was obtained by rupturing whole spinach ( Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl 2 buffered with Tricine at pH 8.5. This CO 2 assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO 2 fixation by MgP were similar to those found with whole chloroplasts. 相似文献
3.
Addition of millimolar sodium glyoxylate to spinach ( Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of 14CO 2 under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO 2 concentrations. Incorporation of 14C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [ 14C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO 2 uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased 14C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased 14CO 2 labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net 14CO 2 fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase 14CO 2 incorporation by 15% in isolated mesophyll cells under air levels of CO 2. 相似文献
4.
A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach ( Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg 2+ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl 2 and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl 2, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent Km (NADH) was 74 micromolar and Km (NADPH) was 81 micromolar. Apparent Vmax was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide. 相似文献
5.
Addition of exogenous Mg 2+ (2 millimolar) to illuminated intact spinach ( Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K +. Addition of K + (10-50 millimolar) reversed both stromal acidification and K + efflux from the chloroplast caused by Mg 2+. These data suggested that Mg 2+ induced reversible H +/K + fluxes across the chloroplast envelope. Ca 2+ and Mn 2+ (2 millimolar) were as effective as 4 millimolar Mg 2+ in causing K + efflux from chloroplasts and inhibition of O 2 evolution. In contrast, 10 millimolar Ba 2+ induced only a small amount of inhibition. The lack of strong inhibition by Ba 2+ indicated that the effects of divalent cations such as Mg 2+ cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg 2+ was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O 2 evolution (43%) induced by Mg 2+. 相似文献
6.
Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach ( Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO 2 and Mg 2+ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO 2 and Mg 2+. 相似文献
7.
Low concentrations (0.5-10 μ m) of antimycin A were shown to increase the rate of CO 2 fixation, O 2 evolution and inorganic phosphate esterification in intact spinach ( Spinacia oleracea) chloroplasts. The increase was highest when the light intensity was saturating. Stimulation was independent of the bicarbonate concentration and was accompanied by an enhancement in the synthesis of glycerate 3-phosphate with a decrease in dihydroxyacetone phosphate. The antibiotic decreased the Michaelis constant of the chloroplast but not of ribulose 1,5-diphosphate carboxylase for bicarbonate. It was suggested that antimycin A is affecting that portion (outer envelope) of the intact chloroplast which contains the enzyme mechanism for controlling the pace of CO 2 fixation. 相似文献
8.
Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, −1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at −1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, −2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to −1.8 megapascals and the chloroplasts had higher rates of CO 2-dependent O 2 evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations. Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20°C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20°C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20°C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [14C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [14C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol. It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology. 相似文献
9.
Phosphoglucomutase (PGM) activity was measured in spinach ( Spinacia oleracea L.) chloroplasts. Initial enzyme activity in a chloroplast lysate was 5 to 10% of total activity measured with 1 micromolar glucose 1,6-bisphosphate (Glc 1,6-P 2) in the assay. Initial PGM activity increased 2- to 3-fold when chloroplasts were illuminated for 10 minutes prior to enzyme measurement and then decreased slowly in the dark. Measurements of total enzyme activity were unchanged by prior light treatment. Initial PGM activity from light treated chloroplasts was sufficient to account for in vivo rates of starch synthesis. Changes in PGM activity were affected by stromal pH and orthophosphate concentration. Photosynthetic inhibitors, dl-glyceraldehyde, glycolaldehyde, and glyoxylate, decreased and 3-phosphoglyceric acid increased light induced changes of PGM activity. Dark preincubation of chloroplasts with 10 millimolar dithiothreitol had no effect upon initial PGM activity, suggesting that light effects did not involve a sulfhydryl mechanism. Hexose monophosphate levels increased in illuminated chloroplasts. Activation of PGM in a chloroplast lysate by Glc 1,6-P 2 was maximal between pH 7.5 and 8.5. Stromal concentrations of Glc 1,6-P 2 were between 20 and 30 micromolar for both light and dark incubated chloroplasts and these levels should saturate PGM activity. Light dependent alterations of enzyme activity may be due to changes of phosphorylated PGM levels in the stroma or are the result of changes in residual activity by the dephosphorylated form of the enzyme. The above results indicate that PGM activity in spinach chloroplasts may be regulated by light, stromal pH, and Glc 1,6-P 2 concentration. 相似文献
10.
The incorporation of 14CO 2 into glycolate by intact spinach leaf ( Spinacia oleracea L. var. Kyoho) chloroplasts exposed to 14CO 2 (NaH 14CO 3, 1 millimolar) in the light was determined as a function of O 2 concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent Km (O 2) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic 14CO 2 fixation under an O 2 atmosphere. 相似文献
11.
Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO 2 was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O 2 evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O 2 and fixed 21 micromoles of CO 2 per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO 2 in air for 24 hours. On low CO 2 the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations. 相似文献
12.
Scenedesmus cells grown on high CO 2, when adapted to air levels of CO 2 for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO 2 from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O 2 evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO 2 pump with K 0.5 for O 2 evolution of less than 5 micromolar CO 2 was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO 3− concentrating process between pH 8 to 10 lowered the K 0.5 (DIC) from 7300 micromolar HCO 3− in high CO 2 grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO 3− pump was inhibited by vanadate (K i of 150 micromolar), as if it involved an ATPase linked HCO 3− transporter. The CO 2 pump was formed on low CO 2 by high-CO 2 grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO 3− pump was partially activated on low CO 2 within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO 3− pump at pH 9 had requirements similar to the activation of the CO 2 pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO 2-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO 2 pump and in some algae an additional vanadate-sensitive plasmalemma HCO 3− transporter for a HCO 3− pump. 相似文献
13.
Chloroplastic respiration was monitored by measuring 14CO 2 from 14C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of 14CO 2 evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phospho enolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The Km for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of 14CO 2 was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO 2 evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO 2 evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH 4Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phospho enolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO 2 and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism. 相似文献
14.
Rates of photosynthetic O 2 evolution, for measuring K 0.5(CO 2 + HCO 3−) at pH 7, upon addition of 50 micromolar HCO 3− to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K1(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO 2 uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O 2 evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO 3), and the rate of 14CO 2 fixation with 100 micromolar [ 14C] HCO 3−. Salicylhydroxamic acid inhibition of O 2 evolution and 14CO 2-fixation was reversed by higher levels of NaHCO 3. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO 2 accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related. 相似文献
15.
Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat ( Triticum aestivum) have rates of photosynthesis as measured by light-dependent O 2 evolution of about 100 to 150 micromoles of O 2 per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO 3 with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O 2 per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset. Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP. Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO3 and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed. 相似文献
16.
Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline → betaine aldehyde → betaine. Our previous experiments with intact chloroplasts, and in vivo18O 2 labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O 2 and reduced ferredoxin, the stromal fraction from spinach ( Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of 18O from 18O 2 by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea ( Pisum sativum L.), a species that does not accumulate betaine. The spinach choline-oxidizing enzyme was stimulated by 10 millimolar Mg 2+, had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin. 相似文献
17.
The respiratory capacity of isolated spinach ( Spinacia oleracea L.) chloroplasts, measured as the rate of 14CO 2 evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [ 14C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO 2 evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH 4Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction. 相似文献
18.
The transport of l-[ 14C]glutamine in oat ( Avena sativa L.) and spinach ( Spinacia oleracea L.) chloroplasts was studied by a conventional single-layer and a newly developed stable double-layer silicone oil filtering system. [ 14C]Glutamine was actively transported into oat chloroplasts against a concentration gradient. Metabolite uptake was greatly affected by the endogenous dicarboxylate pools, which could be easily changed by preloading the chloroplast with specific exogenous substrate. Glutamine uptake was decreased by 44 to 75% in oat chloroplasts preloaded with malate, 2-oxoglutarate (2-OG), and aspartate, but increased by 52% in chloroplasts preloaded with l-glutamate. On the other hand, the uptake of the other four dicarboxylates was decreased by 47 to 79% in chloroplasts preloaded with glutamine. In glutamine-preloaded chloroplasts the uptake of glutamine was inhibited only by l-glutamate. The observed inhibition by l-glutamate was competitive with an apparent Ki value of 32.1 millimolar in oat and 6.7 millimolar in spinach chloroplasts. This study indicates that there are two components involved in glutamine transport in chloroplasts. The major component was mediated via a specific glutamine translocator. It was specific for glutamine and did not transport other dicarboxylates except l-glutamate. A K0.5 value of 1.25 millimolar and Vmax of 45.5 micromoles per milligram of chlorophyll per hour were determined for the glutamine translocator in oat chloroplasts. The respective values were 1.0 millimolar and 16.7 micromoles per milligram of chlorophyll per hour in spinach chloroplasts. A three translocator model, involving the glutamine, dicarboxylate, and 2-OG translocators, is proposed for the reassimilation of photorespiratory NH 3 in chloroplasts of C 3 species. In this three-translocator model the additional transport of glutamine into the chloroplast is coupled to the export of glutamate via the glutamine translocator. This is an extension of the two-translocator model, involving the dicarboxylate and 2-OG translocators, proposed for spinach chloroplasts, (KC Woo, UI Flügge, HW Heldt 1987 Plant Physiol 84: 624-632). 相似文献
19.
Because glyoxylate inhibits CO 2 fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization. The fixation of CO2 by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO2 fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO2 fixation seems to be a general effect of salts of weak acids. Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP+) from intact chloroplast preparations had an apparent Km (glyoxylate) of 140 micromolar and a Vmax of 3 micromoles per minute per milligram chlorophyll. 相似文献
20.
The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO 2. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO 2 results in a gradual inactivation of the RuBP car?ylase. In the presence of CO 2 the initial activity is preserved or increased. CO 2 is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO 2. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO 2 of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO 2 for half activation, while in the light it required 24 μ m CO 2. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg 2+ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO 2 assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO 2 and light-induced changes in the chloroplast stroma such as pH, Mg 2+, and intermediates of the photosynthetic carbon cycle. 相似文献
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