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.

Identification of a potential redox-sensitive interdomain disulfide in the sedoheptulose bisphosphatase of Chlamydomonas reinhardtii.

In the stimulated three-dimensional structure of the Chlamydomonas reinhardtii sedoheptulose bisphosphatase (EC 3.1.3.37) there are two cysteine residues close enough to one another to form a redox-sensitive disulfide bond which would cross-link the nucleotide and carbon substrate domains. Examination of the redox modulation of this sedoheptulose bisphosphatase confirms that it resembles the higher plant enzyme in being activated by reduction. In the wheat and Arabidopsis enzymes, for which there is sequence information and which, like the Chlamydomonas enzyme, can be modeled, both redox-sensitive Cys residues appear to be located on the regulatory nucleotide-binding domain. Apparently different Cys residues are involved in modulation in the algal and higher plant sedoheptulose bisphosphatases.

Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-.

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (I. The Activation of Phosphoribulosekinase and Glucose-6-Phosphate Dehydrogenase Is Relative to the Photosynthetic Supply of Electrons).

Extraction of Chlamydomonas reinhardtii CW-15 cells by rapid freezing and thawing demonstrates that the in vivo activity of the algal glucose-6-phosphate dehydrogenase (G6PDH) is inhibited by the presence of light and activated in the dark, whereas phosphoribulosekinase (PRK) is light activated and inhibited in the dark. The effects of darkening are reversed by incubation with dithiothreitol (DTT) and mimicked by chemical oxidants, indicating that, as in higher plants, reduction via the ferredoxin-thioredoxin system likely regulates these enzymes. The two enzymes varied in their sensitivity to reduction; the inclusion of 0.5 mM DTT during extraction inhibited G6PDH, whereas PRK required treatment with 40 mM DTT for 1 h to reach maximum activation. The activation change for both enzymes was nearly complete within the 1st min after cells were transferred between light and dark, but the level of activation was relative to the incident light at low intensities; G6PDH activity decreased with increasing light, whereas PRK became more active. The reductive inhibition of G6PDH saturated at very low light, whereas PRK activation kinetics closely followed the increase in photosynthetic oxygen evolution. These results indicate that light-driven redox modulation of G6PDH and PRK is more than an on/off switch, but acts to optimize the reduction and oxidation of carbon in the chloroplast in accordance with the supply of electrons.

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.

Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO(3) but Not NH(4) by a Green Alga.

Addition of NO(3) (-) to N-limited Selenastrum minutum during photosynthesis resulted in an immediate drop in the NADPH/NADP ratio and a slower increase of the NADH/NAD ratio. These changes were accompanied by a rapid decrease in glucose-6-phosphate and increase in 6-phosphogluconate, indicating activation of glucose-6-phosphate dehydrogenase and a role for the oxidation pentose phosphate pathway during photosynthetic NO(3) (-) assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH(4) (+) were not consistent with a stimulation of the oxidative pentose phosphate pathway.

Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum.

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO(3) (-) or NH(4) (+) assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH(4) (+) was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO(3) (-) addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO(3) (-) assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO(3) (-) assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH(4) (+) assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO(3) (-) assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.

Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii.

The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii.

The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components.

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii : Identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components.

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

Activation of a chloroplast type of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents.

A chloroplast type of fructose-1,6-bisphosphatase, a central regulatory enzyme of photosynthetic carbon metabolism, has been partially purified from Chlamydomonas reinhardtii. Unlike its counterpart from spinach chloroplasts, the algal FBPase showed a strict requirement for a dithiol reductant irrespective of Mg2+ concentration. The enzymes from the two sources resembled each other immunologically, in subunit molecular mass and response to pH. In the presence of dithiothreitol, the pH optimum for both the algal and spinach enzymes shifted from 8.5 to a more physiologic value of 8.0 as the Mg2+ concentration was increased from 1 to 16 mM. At 1 mM Mg2+, a concentration estimated to be close to physiological, the Chlamydomonas FBPase was active only in the presence of reduced thioredoxin and was most active with Chlamydomonas thioredoxin f. Under these conditions, the enzyme showed a pH optimum of 8.0. The data suggest that the Chlamydomonas enzyme resembles its spinach counterpart in most respects, but it has a stricter requirement for reduction and less strict reductant specificity. A comparison of the properties of the FBPases from Chlamydomonas and spinach will be helpful for elucidating the mechanism of the reductive activation of this enzyme.