Consequences of the presence of 24-epibrassinolide, on cultures of a diatom, Asterionella formosa

Addition of the plant hormone 24-epibrassinolide to culture media stimulated the growth of a freshwater diatom, Asterionella formosa. The hormone stimulated activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme from Calvin cycle, by 6-fold. Other key metabolic enzymes, phosphofructokinase and malate dehydrogenase were also stimulated but to a lesser extent. The activity of glucose-6-phosphate dehydrogenase, involved in the oxidative pentose phosphate pathway, also increased in the presence of the hormone but only under non reducing conditions. In cells stimulated by epibrassinolide, activated enzymes were sensitive to oxidized-DTT. GAPDH purified from cells grown in the presence of the hormone was not associated with a small protein of 8.5 kDa shown to be similar to CP12. Consequently the activity of GAPDH was no longer regulated by either oxidizing or reducing conditions. Among enzymes that, like GAPDH, responded positively to reducing agent were fructose-1,6-bisphosphatase (FBPase) and glucose-6-phosphate dehydrogenase (G6PDH). These enzymes were also sensitive to, and were negatively regulated by, oxidized-DTT. The activities in extracts from illuminated cells differed from those from darkened cells: FBPase, G6PDH and GAPDH, that were activated by DTT in darkened cells were no more activated in illuminated cells, but were oxidized by oxidized-DTT. Thus, oxidizing or reducing conditions mimic the conditions in dark and light, respectively. Unlike the other enzymes, phosphofructokinase (PFK) was inhibited by DTT but oxidized-DTT reversed this effect. The enzymes shown to be redox regulated in vitro by reduction/oxidation are very likely candidates for regulation in vivo by thioredoxins.     

Deactivation of Glucose-6-Phosphate Dehydrogenase by Light-Reduced Thioredoxin

The activity of glucose-6-phosphate dehydrogenase (G6PDH, E. C. 1.1.1.49) in a reconsituted pea chloroplast system was assayed spectrophotometrically by the reduction of NADP, ming glucose-6-phosphate as substrate. Deactivation of G6PDH could be intensified by adding lightreduced thioredoxin (Td) into the reconstituted chloroplast system. The experimental results presented suggest that Td plays an important role not only in the dark activation, but also in the light deactivation of G6PDH in chloroplasts. There were two isozymes of G6PDH in green and in etiolated pea seedlings. The effects of dithiothreitol (DTT) and Td on G6PDH in etiolated seedlings were different from that in chloroplasts. The light regulation of G6PDH in chloroplasts is mediated through Td.     

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (II. Redox Modulation Activates the Oxidative Pentose Phosphate Pathway during Photosynthetic Nitrate Assimilation)

The onset of photosynthetic NO3- assimilation in N-limited Chlamydomonas reinhardtii increased the initial extractable activity of the glucose-6-phosphate dehydrogenase (G6PDH), the key regulatory step of the oxidative pentose phosphate pathway. The total activated enzyme activity did not change upon NO3- resupply. The higher activity, therefore, represents activation of existing enzyme. No activation occurred during NH4+ assimilation. Incubation of extracts with DTT reversed the NO3- stimulation of G6PDH activity, indicating that the activation involved redox modulation of G6PDH. Phosphoribulosekinase, an enzyme activated by thioredoxin reduction, was inhibited at the onset of NO3- assimilation. A 2-fold stimulation of O2 evolution and a 70% decrease in the rate of photosynthetic CO2 assimilation accompanied the enzyme activity changes. There was an immediate drop in the NADPH and an increase in NADP upon addition of NO3-, whereas NH4+ caused only minor fluctuations in these pools. The response of C. reinhardtii to NO3- indicates that the oxidative pentose phosphate pathway was activated to oxidize carbon upon the onset of NO3- assimilation, whereas reduction of carbon via the reductive pentose phosphate pathway was inhibited. This demonstrates a possible role for the Fd-thioredoxin system in coordinating enzyme activity in response to the metabolic demands for reducing power and carbon during NO3- assimilation.     

光还原的硫氧还蛋白对6—磷酸葡萄糖脱氢酶的钝化作用

测定了豌豆(Pisum sativum)幼苗的重组叶绿体中光还原的硫氧还蛋白(Td)对6-磷酸葡萄糖脱氢酶(G6PDH)的钝化作用.结果表明,Td在叶绿体G6PDH的光抑制和暗激活中均起重要的调节作用.在其绿色叶片和黄化组织中,G6PDH都存在着两种同工酶,但二硫苏糖醇(DTT)和Td对黄化幼苗中G6PDH活性的影响与叶绿体的明显不同,DTT对黄化幼苗G6PDH的钝化作用和氧化Td的活化作用均低于对叶绿体中的这两种作用.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.     

Multiple enzyme forms of glyceraldehyde-3-phosphate dehydrogenase in Pseudomonas aeruginosa PAO

Both NAD- and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (EC 1.2.1.12) activities were detected in glucose-grown cells of Pseudomonas aeruginosa strain PAO. After growth on gluconeogenic substrates such as citrate, the activity of the NAD-G3PDH was reduced severalfold in contrast to little change for the NADP-G3PDH. The two G3PDH activities could be separated by ammonium sulphate fractionation. PAGE revealed the presence of two G3PDH isoenzymes of 140 (NADP-specific) and 315 (NAD-specific) kDa. Slight differences were observed in the thermostabilities and pH optima of the two enzymes whereas the regulation of their activities by various compounds varied strongly. The NADP-G3PDH enzyme was activated by ATP, reduced NAD, and fructose 6-phosphate. It was inhibited by fructose 1,6-diphosphate and 6-phosphogluconate. The NAD-G3PDH enzyme was inhibited by ATP, reduced NAD, and 6-phosphogluconate; it was slightly activated by reduced NADP. The possible roles of these isoenzymes in the control of hexose catabolism and gluconeogenesis in P. aeruginosa are discussed.     

Nitrate Reductase and Nitrite Reductase Activity in Dark-Grown Radish Seedlings: Relation to the Supply of NADPH

Functioning of nitrate reductase and nitrite reductase was measured in intact cotyledons from radish seedlings (Raphanus sativus L.) grown in the dark in a nitrate medium. Reduction of nitrate to nitrate did proceed during the whole period of 45 h, whereas the reduction of nitrite in the intact cotyledons dropped abruptly between 20 and 23 h after exposing the roots to nitrate. The activity of the enzymes glucose-6-P dehydrogenase (G6PDH) and 6-P-gluconate dehydrogenase (6PGDH), measured in cotyledon extracts, showed a sharp decline simultaneously with the drop in nitrite reductase activity of the intact cotyledons. It was concluded that the amount of NADPH generated by the enzymes G6PDH and 6PGDH is not sufficient to allow continuous functioning of nitrite reductase after 20 h in cotyledons of seedlings grown in the dark. Therefore, the results from our experiments point to the functioning of nitrite reductase as the rate limiting step in the reduction pathway of nitrate in the dark.     

Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts.

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP- GAPDH-A2B2) involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP-GAPDH and PRK and (b) of only NADP-GAPDH, respectively, in darkened chloroplasts. While the 550-kDa PRK/GAPDH/CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation with dithiothreitol and the effector 1,3-bisphosphoglycerate. In the light, PRK is therefore completely in its activated state under all conditions, even in low light, while GAPDH activation in the light is characterized by a two-step mechanism with 60-70% activation under most conditions in the light, and the activation of the remaining 30-40% occurring only when 1,3-bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP-GAPDH, some of the PRK reaggregates. Recombinant GapA/GapB constructs form the A8B8 complex immediately upon expression in E. coli.     

Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solatium tuberosum L.)

Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) has been purified from potato tuber at least 850-fold to apparent homogeneity as judged by SDS-PAGE. The enzyme was characterized by K_m values of 260 μM for glucose-6-phosphate and 6 μM for NADP and a broad pH optimum between phi 7.5 and 9. NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity. Dithiothreitol (DTT) did not inactivate the enzyme. A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots. A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh-specific primers. A full-length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources. The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%). The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox-independent cytosolic isoform was obtained.     

Difference in activation capacity between oocytes of Pleurodeles waltl matured in vivo and in vitro.

Oocytes of Pleurodeles waltl were activated after in vivo maturation by needle pricking or electric shock. After in vitro maturation, the oocytes were not activated by these stimuli. Coelomic oocytes and the oocytes which began their maturation in vivo could be activated by electric shock. During in vivo oocyte maturation, the activity of glucose-6-phosphate dehydrogenase (G6PDH), the key enzyme of the pentose phosphate cycle, increased while that of phosphofructokinase, the key enzyme of glycolysis, remained unchanged. During progesterone-induced in vitro oocyte maturation, the activity of both enzymes remained unchanged. Oocytes of Misgurnus fossilis matured in vivo and in vitro were activated spontaneously. No changes in the activity of G6PDH were observed during their maturation. These results suggest a relationship between G6PDH activity in the oocyte and oocyte capacity for activation by needle pricking or electric shock.     

Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits

The subcellular distribution and activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) were studied in developing peach (Prunus persica L. Batsch cv. Zaoyu) fruit. Fruit tissues were separated by differential centrifugation at 15,000g into plastidic and cytosolic fractions. There was no serious loss of enzyme activity (or activation) during the preparation of fractions. G6PDH activity was found in both the plastidic and cytosolic compartments. Moreover, DTT had no effect on the plastidic G6PDH activities, that is, the redox regulatory mechanism did not play an important role in the peach fleshy tissue. Results from the immunogold electron-microscope localization revealed that G6PDH isoenzymes were mainly present in the cytosol, the secondary wall and plastids (chloroplasts and chromoplasts), but scarcely found in the starch granules or the cell wall. In addition to a decrease in fruit firmness, the G6PDH activity in the cytotolic and plastidic fractions increased, and anthocyanin started to accumulate during fruit maturation. These results suggest that G6PDH, by providing precursors for metabolic processes, might be associated with the red coloration that occurs in peach fruit.     

Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.     

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10^-4 M) causes a 90% inhibition of CO₂-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [¹⁴C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of ¹⁴CO₂ from added [¹⁴C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG ADP-glucose - DHAP dihydroxyacetone phosphate - DTT dithiothreitol - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HMP hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate) - 6-PGI 6-phosphogluconate - PMP pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate) - RuBP ribulose-1,5-bisphosphate - S7P sedoheptulose-7-phosphate - SBP sedoheptulose-1,7-bisphosphate Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday     

甜杨6-磷酸葡萄糖脱氢酶在抗冻性低温诱导中的作用

对-20℃低温锻炼及脱锻炼过程中甜杨(Populus suaveolens)幼苗的G6PDH、SOD和POD活性、MDA含量和半致死温度(LT50)进行了测定和分析.结果发现,低温锻炼在一定程度上提高了幼苗6-磷酸葡萄糖脱氢酶(G6PDH)、SOD和POD活性,降低了MDA含量和幼苗半致死温度(LT50).另外,将幼苗放回常温(脱锻炼)2 d能引起幼苗的G6PDH、SOD和POD活性的显著下降,并使LT50和MDA含量的迅速回升.结果表明,低温锻炼中G6PDH活性的增加有助于SOD和POD活性的提高,进而对幼苗的LT50和MDA含量的降低有明显的促进作用,G6PDH可能参与了SOD和POD活性的调节和抗冻性的低温诱导.     

ACTIVATION OF HEXOSE MONOPHOSPHATE PATHWAY IN BRAIN BY ELECTRICAL STIMULATION IN VITRO

Abstract— The incubation of cerebral cortical slices for 15 min in Krebs-Ringer-tris (pH 7.6) solution at 37°C with [1-¹⁴C]glucose or [6-¹⁴C]glucose as substrates yielded a C-1:C-6 ¹⁴CO₂ ratio of 1.21, whereas this ratio increased to 3.01 after the application of electrical stimulation (ES). Tissue levels of 6-phosphoglu-conate (6PG) and glucose 6-phosphate (G6P), intermediary metabolites of hexose monophosphate (HMP) pathway, were 7 and 180 nmol/g tissue following 15 min incubation, and increased by 33 and 45 per cent respectively following the application of ES. Activities of 6-phosphogluconate dehydrogenase (6PGDH, 6-phospho- d -gluconate: NADP⁺ 2-oxidoreductase, EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (G6PDH, d -glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49), important enzymes in regulating the activity of HMP pathway, in cerebral cortical slices were 689 and 907 pmol/mg protein/min and were increased by 66 and 25 per cent respectively by the application of ES. Synaptosomal G6PDH and 6PGDH activities were maximally activated by the addition of 40 m m -Na⁺ to the reaction mixture, whereas no activation by Na⁺ was observed in microsomal G6PDH and 6PGDH. Amobarbital inhibited more strongly the Embden–Meyerhof (EM) pathway than the HMP pathway, while imipramine had a stronger inhibitory effect on HMP pathway than on EM pathway in the electrically stimulated cerebral tissues.
The present results indicate that the HMP shunt pathway in the cerebral cortex is activated by the application of ES in vitro , possibly at synaptic regions and may play an important metabolic and functional role in the brain.     

Role of hydrogen peroxide in regulating glucose-6-phosphate dehydrogenase activity under salt stress

The role of hydrogen peroxide in the regulation of glucose-6-phosphate dehydrogenase (G6PDH) activity in the red kidney bean (Phaseolus vulgaris L.) roots under salt stress (100 mM NaCl) was investigated. Salt stress caused the increase of the activities of G6PDH and antioxidative enzymes including ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), as well as H₂O₂ production. The application of H₂O₂ (1 mM) also enhanced the activities of G6PDH as well as antioxidative enzymes. In the presence of exogenous CAT, H₂O₂ content was decreased, and the enhanced activities of G6PDH and antioxidative enzymes induced by NaCl or by exogenous H₂O₂ were also abolished, suggesting that the enhancement of the above enzyme activities under salt stress was a result of the increased endogenous H₂O₂ levels. Further results showed that the effects of NaCl and H₂O₂ on the activities of antioxidative enzymes were diminished by Na₃PO₄ (a G6PDH inhibitor), suggesting G6PDH activity is required in enhancing the activities of antioxidative enzymes. The enhanced membrane leakage, lipid peroxidation, H₂O₂ and O₂ — contents, G6PDH and antioxidative enzyme activities under salt stress were all recovered to control level when the red kidney bean seedlings treated with 100 mM NaCl for 6 d were transferred to the control conditions for 8 d.     

Regulation of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

Activation of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3-phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3-phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time-course of activation appears to depend upon the actual concentration of 1,3-bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The K_a values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the K_a value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP-GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered K_avalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP-GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.