NADP-glyceraldehyde 3-phosphate Dehydrogenase Research Articles

Validation of an Enzyme-Driven Model Explaining Photosynthetic Rate Responses to Limited Nitrogen in Crop Plants.

The limited availability of nitrogen (N) is a fundamental challenge for many crop plants. We have hypothesized that the relative crop photosynthetic rate (P) is exponentially constrained by certain plant-specific enzyme activities, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-G3PDH), 3-phosphoglyceric acid (PGA) kinase, and chloroplast fructose-1,6-bisphosphatase (cpFBPase), in Triticum aestivum and Oryza sativa. We conducted a literature search to compile information from previous studies on C3 and C4 crop plants, to examine the photosynthetic rate responses to limited leaf [N] levels. We found that in Zea mays, NADP-malic enzyme (NADP-ME), PEP carboxykinase (PCK), and Rubisco activities were positively correlated with P. A positive correlation was also observed between both phosphoenolpyruvate carboxylase (PEPC) and Rubisco activity with leaf [N] in Sorghum bicolor. Key enzyme activities responded differently to P in C3 and C4 plants, suggesting that other factors, such as leaf [N] and the stage of leaf growth, also limited specific enzyme activities. The relationships followed the best fitting exponential relationships between key enzymes and the P rate in both C3 and C4 plants. It was found that C4 species absorbed less leaf [N] but had higher [N] assimilation rates (A rate) and higher maximum photosynthesis rates (Pmax), i.e., they were able to utilize and invest more [N] to sustain higher carbon gains. All C3 species studied herein had higher [N] storage (Nstore) and higher absorption of [N], when compared with the C4 species. Nstore was the main [N] source used for maintaining photosynthetic capacity and leaf expansion. Of the nine C3 species assessed, rice had the greatest Pmax, thereby absorbing more leaf [N]. Elevated CO2 (eCO2) was also found to reduce the leaf [N] and Pmax in rice but enhanced the leaf [N] and N use efficiency of photosynthesis in maize. We concluded that eCO2 affects [N] allocation, which directly or indirectly affects Pmax. These results highlight the need to further study these physiological and biochemical processes, to better predict how crops will respond to eCO2 concentrations and limited [N].

Nitric oxide and hydrogen sulfide modulate the NADPH-generating enzymatic system in higher plants.

Nitric oxide (NO) and hydrogen sulfide (H2S) are two key molecules in plant cells that participate, directly or indirectly, as regulators of protein functions through derived post-translational modifications, mainly tyrosine nitration, S-nitrosation, and persulfidation. These post-translational modifications allow the participation of both NO and H2S signal molecules in a wide range of cellular processes either physiological or under stressful circumstances. NADPH participates in cellular redox status and it is a key cofactor necessary for cell growth and development. It is involved in significant biochemical routes such as fatty acid, carotenoid and proline biosynthesis, and the shikimate pathway, as well as in cellular detoxification processes including the ascorbate-glutathione cycle, the NADPH-dependent thioredoxin reductase (NTR), or the superoxide-generating NADPH oxidase. Plant cells have diverse mechanisms to generate NADPH by a group of NADP-dependent oxidoreductases including ferredoxin-NADP reductase (FNR), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), NADP-dependent malic enzyme (NADP-ME), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and both enzymes of the oxidative pentose phosphate pathway, designated as glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). These enzymes consist of different isozymes located in diverse subcellular compartments (chloroplasts, cytosol, mitochondria, and peroxisomes) which contribute to the NAPDH cellular pool. We provide a comprehensive overview of how post-translational modifications promoted by NO (tyrosine nitration and S-nitrosation), H2S (persulfidation), and glutathione (glutathionylation), affect the cellular redox status through regulation of the NADP-dependent dehydrogenases.

Diurnal temperature-related variations in photosynthetic enzyme activities of two C<sub>4</sub> species of Chenopodiaceae grown in natural environment

The effects of the diurnal variations in ambient temperature on some C3 and C4 enzymes in the Salsola dendroides and Suaeda altissima species of Chenopodiaceae family were studied during the intensive vegetation period. Activities of phosphoenolpyruvate carboxylase (PEPC) and cytosolic aspartate aminotransferase (AsAT) were shown to decrease in both species in the afternoon and evening. The activity of the mitochondrial AsAT decreased in S. altissima, remained relatively constant in S. dendroides during the day. The activity of alanine aminotransferase was high in the S. dendroides species in the morning and evening and decreased in the S. altissima species by the evening. Glucose-6-phosphate activated PEPC in both species throughout the day. The study of the redox status-regulated C3 enzymes showed temperature-related increases in NADP-glyceraldehyde 3-phosphate dehydrogenase activity in both plants, in fructose-2,6-bisphosphatase activity in the S. altissima species, and in NADP-MDH activity in the S. dendroides species in the afternoon.

Glycolytic enzyme activities and gene expression in Cicer arietinum exposed to water-deficit stress.

The specific activities and transcript levels of glycolytic enzymes were examined in shoots of chickpea (Cicer arietinum L.) cultivars, Pusa362 (drought tolerant) and SBD377 (drought sensitive), subjected to water-deficit stress 30 days after sowing. Water-deficit stress resulted in decrease in relative water content, chlorophyll content, plant dry weight, and NADP/NADPH ratio and increase in NAD/NADH ratio in both the cultivars. A successive decline in the specific activities of fructose-1,6-bisphosphate aldolase (aldolase), 3-phosphoglycerate kinase (PGK), and NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and elevation in the specific activities of phosphoglycerate mutase (PGM) and triosephosphate isomerase (TPI) was observed in both the cultivars under stress as compared to their respective control plants. The specific activities of hexokinase, fructose-6-phosphate kinase (PFK), and NAD-GAPDH were least affected. The transcript levels of PGK and NADP-GAPDH decreased and that of glucose-6-phosphate isomerase (GPI), PGM, and PFK increased in response to water-deficit stress while water-deficit stress had no effect on the steady-state transcript levels of hexokinase, aldolase, TPI, and NAD-GAPDH. The results suggest that under water-deficit stress, the activities and transcript levels of most of the glycolytic enzymes are not significantly affected, except the increased activity and transcript level of PGM and decreased activities and transcript levels of PGK and NADP-GAPDH. Further, the glycolytic enzymes do not show much variation between the tolerant and sensitive cultivars under water deficit.

Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings

Seedlings of sweet orange (Citrus sinensis) were fertilized for 14 weeks with boron (B)-free or B-sufficient (2.5 or 10 microM H(3)BO(3)) nutrient solution every other day. Boron deficiency resulted in an overall inhibition of plant growth, with a reduction in root, stem and leaf dry weight (DW). Boron-starved leaves showed decreased CO(2) assimilation and stomatal conductance, but increased intercellular CO(2) concentrations. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and stromal fructose-1,6-bisphosphatase (FBPase) were lower in B-deficient leaves than in controls. Contents of glucose, fructose and starch were increased in B-deficient leaves while sucrose was decreased. Boron-deficient leaves displayed higher or similar superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activities, while dehydroascorbate reductase (DHAR) and catalase (CAT) activities were lower. Expressed on a leaf area or protein basis, B-deficient leaves showed a higher ascorbate (AsA) concentration, but a similar AsA concentration on a DW basis. For reduced glutathione (GSH), we found a similar GSH concentration on a leaf area or protein basis and an even lower content on a DW basis. Superoxide anion (O(2)(-)) generation, malondialdehyde (MDA) concentration and electrolyte leakage were higher in B-deficient than in control leaves. In conclusion, CO(2) assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. Although B-deficient leaves remain high in activity of antioxidant enzymes, their antioxidant system as a whole does not provide sufficient protection from oxidative damage.

Amount of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) Protein and levels of mRNAs of rbcS and rbcL in the leaves at different positions in transgenic rice plants with decreased content of Rubisco

The differences in the amounts of various photosynthetic components, including ribulose- 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and chlorophyll (Chl), and the activities of chloroplastic fructose-1,6-bisphosphatase (Cp-FBPase), NADP-glyceraldehyde-3- phosphate dehydrogenase (NADP-G3PDH), ribulose-5-phosphate kinase (Ru5P kinase), and sucrose-phosphate synthase (SPS) enzymes were examined in leaf blades at different leaf positions on the main culms of transgenic rbcS antisense rice plants. The transcript levels of rbcS and rbcL mRNAs were also determined. All the leaves were collected when the flag leaves became fully expanded. Although the Rubisco content was much lower in the upper leaves in the rbcS antisense plants than in the wild-type plants, the difference was less pronounced in lower leaves. Whereas the levels of rbcS mRNA in all the leaves were depressed in the antisense plants, the levels of rbcL mRNA did not differ. Both rbcS and rbcL mRNA levels in lower leaves were very low in the wild-type and antisense plants. Chl content and the activities of Cp-FBPase, NADP-G3PDH, Ru5P kinase, and SPS were slightly higher in the antisense plants. However, the changes among the leaves at different positions were similar to those in the wild-type plants. These results suggest that a selectively slow decrease in the Rubisco content in lower leaves occurs in the rbcS antisense plants and that it is caused by the attenuation of degradation and not by the maintenance of Rubisco synthesis.

Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes

The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea (Pisum sativum) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO2 (0.1 mM NaHCO3) than that at optimal CO2 (1.0 mM NaHCO3). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.

Phosphate Starvation Inducible `Bypasses' of Adenylate and Phosphate Dependent Glycolytic Enzymes in Brassica nigra Suspension Cells

When Brassica nigra leaf petiole suspension cells were subjected to 7 days of inorganic phosphate (Pi) starvation the extractable activity of: (a) pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate phosphatase, and phosphoenolpyruvate carboxylase increased at least fivefold, (b) phosphorylating NAD-glyceraldehyde 3-phosphate dehydrogenase decreased about sixfold, and (c) ATP:fructose 6-phosphate 1-phosphotransferase, 3-phosphoglycerate kinase, pyruvate kinase, or NAD malic enzyme was not altered. Pi deprivation also resulted in significant reductions in extractable levels of Pi, ATP, ADP, fructose 2,6-bisphosphate, and soluble protein, but caused a sixfold elevation in free amino acid concentrations. No change in inorganic pyrophosphate concentration was observed following Pi starvation. It is hypothesized that pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion.

Calvin-Benson Cycle Enzymes in Guard-Cell Protoplasts from Vicia faba L

Activities of Calvin-Benson cycle enzymes were found in protoplasts of guard cells from Vicia faba L. The activities of NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD) and ribulose-1,5-bisphosphate carboxylase (RuBPC) were 2670 and 52 micromoles per milligrams chlorophyll per hour, respectively. Activities of NADP-GAPD and RuBPC in guard cells were increased by red light illumination, and the light activations were inhibited completely by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Enzymes related to the Calvin-Benson cycle such as 3-phosphoglycerate kinase (PGAK), triose phosphate (TP) isomerase, and fructose-1,6-bisphosphatase (FBPase) were shown to be present in guard-cell chloroplasts. From these results, we conclude that the photosynthetic carbon reduction pathway is present in guard-cell chloroplasts of Vicia faba. We compared these enzyme activities in guard cells with those in mesophyll cells. The activities of NADP-GAPD and PGAK were more than several-fold higher and that of TP isomerase was much higher in guard-cell chloroplasts than in mesophyll chloroplasts. In contrast, activities of RuBPC and FBPase were estimated to be roughly half of those in mesophyll chloroplasts. High activities of PGAK, NAD-GAPD, and TP isomerase were found in fractions enriched in cytosol of guard cells. Illumination of guard-cell protoplasts with red light increased the cellular ATP/ADP ratio from 5 to 14. These results support the interpretation that guard cells utilize a shuttle system (e.g. phosphoglycerate [PGA]/dihydroxyacetone phosphate [DHAP] shuttle) for an indirect transfer of ATP and reducing equivalents from chloroplasts to the cytosol.

Compartmentation of glucose 6-phosphate dehydrogenase and glyceraldehyde 3-phosphate: NADP reductase in maize leaves

Abstract The distribution of glucose 6-phosphate dehydrogenase and D-glyceraldehyde 3-phosphate: NADP reductase activities in maize leaf blades was investigated by differential tissue fractionation. The reliability of this technique was verified on the basis of distribution of several marker enzymes (NADP-malate dehydrogenase and phosphoenolpyruvate carboxylase for mesophyll cells, NADP-malic enzyme and fructose 1,6-bisphosphate aldolase for bundle-sheath cells). Glucose 6-phosphate dehydrogenase is found mainly (70%) in the mesophyll, similar to the triose phosphate dehydrogenases, both NAD- and NAD(P)-dependent. D-glyceraldehyde 3-phosphate: NADP reductase occurs 75% in the mesophyll. Neither enzyme is found in chloroplasts. Specific activities of nine leaf enzymes were followed for up to 7 days after exposure of 4-day-old dark-grown seedlings to the light. While several activities related to C4 photosynthesis (aldolase, phosphoenolpyruvate carboxylase, NADP-glyceraldehyde 3-phosphate dehydrogenase, NAD...

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 +/- 0.07 micromoles of CO(2) per minute per milligram at 25 degrees C) throughout the experimental period.Kinetic parameters related to CO(2) fixation were examined using the purified carboxylase. The K(m)(CO(2)) and V(max) values were 12 micromolar and 1.45 micromoles of CO(2) per minute per milligram, respectively (pH 8.2 and 25 degrees C). The in vitro specific activity calculated at the atomospheric CO(2) level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.

Light-Dependent Reduction of Hydrogen Peroxide by Ruptured Pea Chloroplasts

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H(2)O(2)-dependent oxidation of ascorbate and ascorbate-dependent reduction of H(2)O(2). The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K(m) value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN(3), KCN, and 8-hydroxyquinoline but not ZnCl(2) or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H), 2 millimolar l-ascorbate, and substrate concentrations of oxidized or reduced glutathione, catalyzed O(2) evolution when H(2)O(2) was added. Since the reaction was not inhibited by 0.1 millimolar NaN(3) and did not occur in the dark, it was concluded that catalase was not involved. Light-plus-H(2)O(2)-dependent O(2) evolution consisted of two distinct phases. The first phase was ascorbate-dependent and typically represented 10% of the total amount of O(2) evolved. The second phase was dependent on ascorbate and glutathione. The properties of the second phase were consistent with the operation of light-coupled glutathione reductase sequentially coupled to glutathione dehydrogenase and ascorbate peroxidase.

Occurrence of cytoplasmic f- and m-type thioredoxins in leaves

There is a growing body of evidence that thioredoxins (proteins that act as regulatory messengers in linking light to enzyme modulation in chloroplasts [ 1,2]) occur in multiple forms in photosynthetic cells [3-61. Two types of thioredoxins are found in chloroplasts (thioredoxins f and m), and a third type is found outside chloroplasts, possibly in the cytoplasm (thioredoxin c) [6]. Chloroplast thioredoxinsfand m are reduced photochemically by chloroplasts via ferredoxin and ferredoxin-thioredoxin reductase or, independently of light, in vitro by the nonphysiological sulfhydryl reagent dithiothreitol. When reduced, thioredoxinf activates specific chloroplast enzymes, including enzymes of the reductive pentose phosphate cycle (fructose-l ,6-bisphosphatase (Fru-Psse), NADPglyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, sedoheptulose-1,7-bisphosphatase [6]) and an enzyme of secondary plant metabolism (phenylalanine ammonia lyase [7]). By contrast, thioredoxin m is specific in its activation of the single chloroplast enzyme, NADP-malate dehydrogenase (NADP-MDH [6]). The function of cytoplasmic thioredoxin c is unknown. In our research, thioredoxin c resembled its chloroplast counterparts in promoting the activation of chloroplast enzymes. However, unlike chloroplast thioredoxins, thioredoxin c appeared to be nonspecific in that when reduced with dithiothreitol it

Ferredoxin/thioredoxin system functions with effectors in activation of NADP-glyceraldehyde 3-phosphate dehydrogenase of barley seedlings

NADP-glyceraldehyde 3-phosphate dehydrogenase, a chloroplast enzyme whose activity has been historically linked to greening, was present in an inactive state in barley seedlings germinated in the dark. The inactive enzyme was converted to an active enzyme by effectors such as ATP and by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with the nonphysiological sulfhydryl reagent dithiothreitol. NAD-linked glyceraldehyde 3-phosphate dehydrogenase was not affected by enzyme effectors or thioredoxin under these conditions. The results suggest that NADP-glyceraldehyde 3-phosphate dehydrogenase is present throughout the life cycle of barley plants. The inactive enzyme is converted during greening to an active state by light either via enzyme effectors or via the ferredoxin-thioredoxin system.

Separation of Mesophyll Protoplasts and Bundle Sheath Cells from Maize Leaves for Photosynthetic Studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and "malic enzyme" (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.

Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase

The development of NADP- and NAD-dependent glyceraldehyde 3-phosphate dehydrogenase and NADH-specific glyoxylate reductase was followed in Sinapis alba cotyledons grown in the dark or under continuous red and far red light. All three enzyme activities are promoted by light, continuous far red light being more than twice as effective as continuous red light. The activities of the NADP-glyceraldehyde 3-phosphate dehydrogenase and glyoxylate reductase increase in the far red light from 36 to 96 hours. They remain constant until at least 120 hours after sowing and are respectively 11 and 6 times higher than the maximum dark activities. Contrary to this, the activity of the NAD-glyceraldehyde 3-phosphate dehydrogenase is scarcely more than doubled under continuous far red irradiation relative to its maximal dark level, and its time course curve is displaced along the time axis, with the activity increasing between 24 and 72 hours after sowing.The increase in activity of NADP-glyceraldehyde 3-phosphate dehydrogenase and glyoxylate reductase is inhibited by d-threo-chloramphenicol but not by the l-threo isomer at concentrations of 200 micrograms per milliliter or less, whereas the slight inhibitory effect of chloramphenicol on the NAD-glyceraldehyde 3-phosphate dehydrogenase is not stereospecific. The three enzyme activities are inhibited by cycloheximide.When Knop's solution is used as growth medium it strongly promotes NADP-glyceraldehyde 3-phosphate dehydrogenase activity in vivo and is twice as effective in the red light as in the far red light and dark. The activity of NAD-glyceraldehyde 3-phosphate dehydrogenase is only slightly and almost equally enhanced by Knop's solution in the dark, red, and far red light.These results are consistent with the following conclusions. [List: see text].

Preparation of cellular plant organelles from spinach leaves

Broken chloroplasts, intact chloroplasts, mitochondria, and microbodies were isolated and purified from spinach leaf tissue using a sequential sedimentation rate-equilibrium density (S-ϱ) centrifugation technique. Organelle pellets (1000 g and 3000 g) were prepared in 0.5 m sucrose, 1 m m EDTA, 1 m m 2-mercaptoethanol, and 0.1% bovine serum albumin all in 0.05 m Tris buffer, pH 7.5. The organelle pellets were separately layered on linear sucrose-density gradients of 40–80% ( w v ) and centrifuged for 3 hr at 25,000 rpm ( ω 2 t = 74 × 10 9 rad 2 · sec −1) in an ultracentrifuge. The 1000 g pellet resolved into broken chloroplasts (without outer envelope and stroma), intact chloroplasts (with outer envelope and stroma) plus mitochondria, and microbodies; the 3000 g pellet resolved into broken chloroplasts, mitochondria, and microbodies. Electron micrographs and marker enzymes revealed a high degree of purity. Markers used were glycolate oxidase and slow particulate malic dehydrogenase for microbodies, cytochrome c oxidase and fast particulate malic dehydrogenase for mitochondria, NADP glyceraldehyde 3-phosphate dehydrogenase and chlorophyll for intact chloroplasts, and chlorophyll for broken chloroplasts. Estimates of purity using the enzyme markers suggested that the intact chloroplast fraction was about 13% contaminated by mitochondria and 3.5% by microbodies. The mitochondrial fraction was contaminated about 12% with microbodies and was essentially free from chlorophyll. The 3000 g microbody fraction was essentially pure. The estimated isopycnic densities for the organelles were: broken chloroplasts, 1.17; intact chloroplasts, 1.21; mitochondria, 1.21; and microbodies, 1.25 g/cm 3. When estimated by centrifugation in a 25–50%. ( w v ) sucrose gradient, the sedimentation-rate orders were: intact chloroplasts > broken chloroplasts > microbodies > mitochondria.