Limited proteolysis of inactive tetrameric chloroplast NADP-malate dehydrogenase produces active dimers

Carboxy-terminal amino acids of NADP-dependent malate dehydrogenase (EC 1.1.1.82) from pea chloroplasts were removed by treatment with carboxypeptidase Y. This results in the activation of the inactive oxidized enzyme, while activation by light in vivo is thought to occur via reduction of an intrasubunit disulfide bridge. After proteolytic activation the oxidized enzyme had a specific activity of 100 U/mg protein, which is 50% of the maximal activity of the control enzyme in the reduced state. When the truncated enzyme was reduced with dithiothreitol (DTT), the specific activity was further increased to 1200 U/mg. While the native enzyme is composed of four identical subunits of 38,900 Da, the truncated malate dehydrogenase forms dimers composed of two subunits of 38,000 Da. No further change of molecular mass or activity was noticed subsequent to prolonged incubation of native NADP-malate dehydrogenase with carboxypeptidase Y for several days. When the enzyme is denatured by 2 M guanidine-HCl, the proteolytic activation proceeds more rapidly, but only transiently. The truncated enzyme is less accessible to activation by reduced thioredoxin, but the stimulation of activity by DTT alone is more rapid than that of the native enzyme. These results indicate that only a small carboxy-terminal peptide of native NADP-malate dehydrogenase from pea chloroplasts is accessible to proteolytic degradation and that this peptide is involved in the regulation of activity, tetramer formation, and thioredoxin binding. While the pH optimum for catalytic activity of the intact reduced enzyme is at pH 8.0-8.5, it is shifted to more acidic values upon proteolysis of NADP-malate dehydrogenase. At pH values below 8 the reduced truncated enzyme exhibits substrate inhibition by oxaloacetate.     

pH Dependence of the Km(CO(2)) of Ribulose 1,5-Diphosphate Carboxylase

The Km(CO(2)) values of ribulose 1,5-diphosphate carboxylase in freshly ruptured spinach (Spinacia oleracea L.) chloroplasts and in the purified form isolated from spinach leaves were found to be pH dependent. Raising the pH of the assay solution produced a substantial decrease in the Km(CO(2)) of both enzyme systems. In freshly ruptured chloroplasts at pH 7.2 the Km(CO(2)) was 25 mum, at pH 8 it decreased to 19 mum, and at pH 8.8 a further decrease to 7 mum was found. With the purified enzyme at pH 7.2 the Km(CO(2)) was 147 mum, while the corresponding Km values for pH 8 and 8.8 were 34 and 15 mum CO(2), respectively. The latter figure approximates the physiological Km(CO(2)) of 10 mum estimated for photosynthesizing leaves and intact chloroplasts. The maximum velocity for both enzyme systems at optimum substrate levels was at pH 8, but the highest calculated rate of CO(2) uptake at atmospheric CO(2) levels occurred at pH 8.8. These results support the proposal that the light-induced efflux of protons out of the chloroplast stroma may be a major factor involved with the reported in vivo light activation of ribulose 1,5-diphosphate carboxylase.     

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.     

Lipid biosynthesis by chloroplasts isolated from developing Zea mays

Fatty acid biosynthesis by isolated plastids has been examined in relation to chloroplast development and differentiation in leaves of maize plants grown in light for 7 days. Biosynthesis of fatty acids from acetate by proplastids prepared from the basal regions of the leaf was low and mainly palmitate was synthesized. The greatly increased utilization of acetate for fatty acid biosynthesis as the plastids increased in size was due to an increased synthesis of oleate. The maximum synthesis of total fatty acids and monoenoic fatty acids was obtained in chloroplasts prepared from leaf tissue 6–8 cm from the base of the plant where granal formation was most active. Fully-developed chloroplasts prepared from distal regions of the leaf were less active in fatty acid biosynthesis. Maize chloroplasts failed to synthesize fatty acids when isolated by methods commonly used to prepare active spinach chloroplasts. The method of isolation which included a density gradient gave a high proportion of Class I chloroplasts from maize leaves and incorporated up to about 10% of the acetate used. Biosynthesis of unsaturated fatty acids, especially with chloroplasts prepared from the most mature tissue, was increased by the addition of both mitochondrial and microsomal fractions. Increases in polyunsaturated fatty acids were also obtained but the proportions in the newly-synthesized fatty acids were well below the endogenous levels. Monoenoic synthesis was greatly stimulated by increasing the pH in the range 7·0–8·0 and also the highest proportions of unsaturated fatty acids were obtained at short incubation times.     

Lipid Synthesis and Ultrastructure of Isolated Barley Chloroplasts

The cell organelle contents of chloroplast preparations made from barley leaves with salt and sucrose isolation media at pH 6 and 8 were determined and compared with the acetate incorporating activity of these preparations. A chloroplast preparation obtained with 0.5 m sucrose at pH 8 gave the highest number of intact chloroplasts (with envelope and stroma), the lowest number of contaminating mitochondria, and the highest activity in light dependent acetate incorporation into lipids. In the preparations observed, the light induced lipid synthesizing capacity correlates well with the percentage of intact chloroplasts. It is suggested that the intact chloroplasts are responsible for the light induced lipid synthesis of the preparations and that the synthesizing enzymes are localized in the chloroplast stroma. Acetate is mainly incorporated into palmitic and oleic acids. The low yield of intact chloroplasts and of light induced lipid synthesis in preparations isolated at pH 6 seem to result from the action of galactolipid lipase(s).     

Equilibrium binding of thioredoxin fB to chloroplastic fructose bisphosphatase. Evidence for a thioredoxin site distinct from the active site

Thioredoxin fB, the protein activator of chloroplastic fructose 1,6-bisphosphatase, strongly binds its target enzyme with a stoichiometry of one protein dimer per enzyme tetramer. The thioredoxin binding site is distinct from the active site and the dissociation constant of the protein-enzyme complex has the extremely small value of 769 nM at pH 7.5. This interaction involves both ionic and hydrophobic contributions and is enhanced by a pH increase from 7 to 8. These results suggest that the above molecular properties may be involved in the light activation of chloroplastic fructose bisphosphatase.     

Alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia.Photophosphorylation was partially inhibited by 100 μM dihydrosanguinarine, 100 μM dihydrochelerythrine (benzophenanthridine alkaloids); 500 μM O,O'-dimethylmagnoflorine, 500 μM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution.Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 μM.     

Photoactivation of the latent O2-evolving center in chloroplasts isolated from dark-grown spruce seedlings

The latent O₂-evolving center in chloroplasts isolated from spruce [Picea abies (L.) Karst.] seedlings grown in the dark was readily activated by pre-illuminating the chloroplast suspension with weak white light. The photoactivation depended on pH with the optimum at pH 7–8, and was strongly stimulated by ascorbic acid. The optimal stimulation was also obtained at pH 7–8. The temperature dependence of the photoactivation suggested the involvement of some dark reaction in the activation process.     

Regulation of NADP-malate dehydrogenase in C4 plants: relationship among enzyme activity, NADPH to NADP ratios, and thioredoxin redox states in intact maize mesophyll chloroplasts

NADP-malate dehydrogenase activity, the ratio of NADPH to NADP, and thioredoxin redox state in Zea mays chloroplasts were determined after various treatments. Following transfer from dark to light, NADP-malate dehydrogenase was activated more than 20-fold within 10 min while the proportion of pyridine nucleotide as NADPH increased from about 25 to 90%, and the proportion of thioredoxin in the reduced form increased from 20 to more than 90%, in less than 1 min. After transfer back to the dark, NADPH levels dropped very rapidly to the initial values recorded before illumination, while enzyme activity and reduced thioredoxin levels decreased more slowly. Addition of oxaloacetate or 3-phosphoglycerate to illuminated chloroplasts results in a decrease of about 70% in the activity of NADP-malate dehydrogenase, a 30% decrease in the level of NADPH, and a 25% decrease in the reduced thioredoxin content. Adding dihydroxyacetone phosphate and pyruvate had no effect. These results are considered in relation to the hypothesis that NADP-malate dehydrogenase activity in chloroplasts may be determined by factors regulating the ratio of NADPH to NADP as well as those influencing the redox state of thioredoxin.     

Ferrochelatase of spinach chloroplasts

Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.     

Selective Formation of Photosystem 1 under Illumination at Low Intensity

Analyses of chlorophylls a and b and P700 in the wheat leaves grown for 8 days under illumination with white light at different intensities suggested selective formation of photosystem 1 of the photosynthesis at low light intensities. This was confirmed for the two types of chloroplasts isolated from leaves grown at light intensities of 1.1 and 240 μ W/cm², respectively, by measuring their pigment compositions, activities of photosystems 1 and 2, and absorption and fluorescence spectra. The chloroplasts developed at the low intensity showed properties only of photosystem 1 while those developed at the high intensity showed properties of both photosystems 1 and 2. Only photosystem 1 particles were obtained by fractionation of low intensity chloroplasts by treatment with digitonin followed by centrifugation, while high intensity chloroplasts could be fractionated into photosystem-1 and photosystem-2 particles. When the leaves grown at low light intensity were illuminated with strong light, photosystem 2 was developed. The fluorescence emission spectrum of low intensity chloroplasts at 77°K showed two peaks at 685 and 734 nm, and the spectrum of high intensity chloroplasts showed three peaks at 685, 697 and 740 nm.     

Changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases (of initial differentiation) in maize calluses and their chloroplasts

Maize calluses and their isolated chloroplasts were analysed to study the changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases of the first day after subculture in maintenance medium (containing 2,4-D) and differentiation medium (without 2,4-D). Free polyamine content changed significantly in both differentiating calluses and chloroplasts showing a maximum during light phase and also increasing after mid-dark phase. Acid-insoluble polyamines showed a similar trend. In whole cells from the callus cultured in maintenance medium, the changes were not significant, except for free putrescine which increased in the dark phase. In chloroplasts of both types of calluses, the trend was similar. Arginine decarboxylase activity in vitro assayed in optimal conditions was not affected by hormone deprivation either in whole cells from the callus or in chloroplasts. The formation of putrescine by arginine decarboxylase activity gradually increased in the light until 9–12 h after subculture, whereas at the onset of the dark phase, a significant decrease was observed. Ornithine decarboxylase activity in vitro always showed slight changes, except in growing callus where putrescine synthesis increased abruptly at 8 h and decreased thereafter. Transglutaminase was immunodetected in whole cells from the callus and in isolated chloroplasts by western blot. In the entire cells, protein substrates were found which were not present in isolated chloroplast. Transglutaminase activity was light sensitive and also affected by hormone deprivation. This enzyme was more active in differentiation than in maintenance medium, in both callus and chloroplasts, in light and dark phases. These data indicate that, the parameters studied here are not only light affected but also regulated by a daily rhythm.     

Effect of {alpha}-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Respiration in isolated mung bean shoot mitochondria was notaffected by either of the tomatine treatments (10^–3 M,pH 5, and 10^–5 M, pH 8) but was reduced in the excisedshoots by both treatments, although only at the higher of thetwo temperatures (5 °C and 25 °C). Inhibition was gradualand took at least 2 h. Tomatine treatment of excised shootsalso resulted in an increased leakage of K+. PS II activityin isolated spinach leaf chloroplasts was reduced only by thehigh pH tomatine treatment at 25 °C. Again, about 2 h treatmentwas required before significant effects were observed but thealkaloid did not cause disintegration of the chloroplast asmeasured by pigment release. Disruption of lysosomes isolatedfrom cauliflower inflorescence tissue and release of acid phosphatasewas enhanced by tomatine. Initially only 10^–5 M tomatineat pH 8 was effective but, later, effects could only be obtainedwith 10^–3 M alkaloid at pH 5. The differential susceptibilityof these organelles to tomatine is discussed in relation tomembrane structure and to the mode of toxicity of the alkaloid.     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

Light activation of fructose bisphosphatase in isolated spinach chloroplasts and deactivation by hydrogen peroxide

Spinach chloroplast fructose bisphosphatase (EC 3.1.3.11.) exists in both oxidised and reduced forms. Only the latter has the kinetic properties that allow it to function at physiological concentrations of fructose 1,6-bisphosphate and Mg²⁺. Illumination of freshly prepared type A chloroplasts causes a conversion of oxidised to reduced enzyme. The rate of this conversion does not limit the rate of CO₂ fixation. In the dark the reduced enzyme partially reverts back to the oxidised form. If catalase is omitted from the reaction medium the rate of CO₂ fixation by chloroplasts is decreased and seems to be limited by the rate of conversion of the enzyme to the reduced form. The physiological significance of the light dependent generation of dithiol compounds (such as thioredoxin) within chloroplasts is discussed.     

Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts.

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP/2 e ratios from the quamdum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9-11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 1/1 ratio, was about 4 both at pH 7.6 ant at 7.0. ATP/2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2. Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chlrooplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP/2e ratios were calculated to be between 1.1 and 1.4. Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 mumoles times mg-1 chlorophyll times h-1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP/2e ratios were obtained under con a decreENG     

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.     

Blue light-modulation of chlorophyll a fluorescence transients in guard cell chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO₂ concentration on the fluorescence transients was investigated in the presence of HCO₃⁻ at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO₂ concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.     

Biophysical studies on subcellular particles V. Effects of temperature on the ferricyanide-Hill reaction, the light-induced pH shift and the light scattering response of isolated spinach chloroplasts

Effects of temperature on the ferricyanide-Hill reaction, thelight-induced pH shift (JpH) and the light scattering response(4LS) of isolated spinach chloroplasts were studied at temperaturesranging from 5 to 60°C. Activities which produce JpH and JLS under actinic illuminationdecreased with a rise in the reaction temperature above 30°Cand disappeared at 50°C. The ferricyanide-Hill reactionat pH 6.0 was stimulated by raising the reaction temperaturefrom 5 to 35°C, was slowed at higher temperatures and wasinactivated at 50°C. 4LS activity at temperatures below20°C depended on the activity of electron transport. With a rise in temperature of a suspension of chloroplasts whichexhibited JLS under actinic illumination, the scattering intensitydecreased. The decrease in scattering intensity was limited,at most, to an extent equal to ALS. The behavior of JLS whichdiffered from that of basal scattering is discussed in connectionwith a mechanism of JLS formation other than the shrinkage-swellingcycle. When activities were measured at 25°C with chloroplastswarmed transiently (2 min) at various temperatures (25–60°C),the dependence of JLS on the wanning temperature was identicalwith that on the reaction temperature, while the activitiesof both ferricyanide reduction and JpH formation were higherthan those measured at the corresponding reaction temperature.This suggested that the mechanism for 4LS formation could beirreversibly altered by a warming treatment and that it wasless stable against heat than the mechanisms of ferricyanidephotoreduction and 4pH formation. That is, transiently warmedchloroplasts showed a similar behavior to alkane-treated chloroplasts.The similarity is discussed in relation to the activity changethrough the possible and common cause of disordering of theorganized structures of lipophilic components in the lamellarmembranes. (Received June 5, 1971; )     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.