Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.     

Carbon starvation mediated changes in carbohydrate metabolism inNeurospora crassa

Carbon starvation conditions were found to increase the activities of gluconeogenic enzymes such as malic enzyme, cytosolic malate dehydrogenase and isocitrate lyase along with proteases and inhibition in glucose catabolic enzymes such as G6P dehydrogenase and FDP aldolase inNeurospora crassa     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, ¹³C metabolic flux analysis with parallel studies on [1-¹³C^Fru]sucrose, [1-¹³C^Glc]sucrose, and [¹³C₆^Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS^Man or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Ethanol Production by Thermophilic Bacteria: Relationship Between Fermentation Product Yields of and Catabolic Enzyme Activities in Clostridium thermocellum and Thermoanaerobium brockii

Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H₂ and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H₂ addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent K_m, V_max, and Q₁₀ values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H₂ production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.     

Photosynthesis in Rhodospirillum rubrum. III. Metabolic Control of Reductive Pentose Phosphate and Tricarboxylic Acid Cycle Enzymes

Enzymes of the reductive pentose phosphate cycle including ribulose-diphosphate carboxylase, ribulose-5-phosphate kinase, ribose-5-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose-1,6-diphos-phatase were shown to be present in autotrophically grown Rhodospirillum rubrum. Enzyme levels were measured in this organism grown photo- and dark heterotrophically as well. Several, but not all, of these enzymes appeared to be under metabolic control, mediated by exogenous carbon and nitrogen compounds. Light had no effect on the presence or levels of any of these enzymes in this photosynthetic bacterium.

The enzymes of the tricarboxylic acid cycle and enolase were shown to be present in R. rubrum cultured aerobically, autotrophically, or photoheterotrophically, both in cultures evolving hydrogen and under conditions where hydrogen evolution is not observed. Light had no clearly demonstrable effect on the presence or levels of any of these enzymes.

     

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Construction of a constitutively expressed homo-fermentative pathway in Lactobacillus brevis

Lactobacillus brevis is a promising lactic acid producing strain that simultaneously utilizes glucose and xylose from lignocellulosic hydrolysate without carbon catabolic repression and inhibition. The production of by-products acetic acid and ethanol has been the major drawback of this strain. Two genes, pfkA (fructose-6-phosphate kinase [PFK]) and fbaA (fructose-1,6-biphosphate aldolase [FBA]), that encode the key enzymes of the EMP/glycolytic pathway from Lactobacillus rhamnosus, were fused to the downstream of the strong promoter P32 and expressed in L. brevis s3f4 as a strategy to minimize the formation of by-products. By expressing the two enzymes, a homo-fermentative pathway for lactic acid production was constructed. The lactic acid yields achieved from glucose in the transformants were 1.12 and 1.16 mol/mol, which is higher than that of the native strain (0.74 mol/mol). However, the lactic acid yield from xylose in the transformants stayed the same as that of the native strain. Enzyme assay indicated that the activity of the foreign protein FBA in the transformants was much higher than that of the native strains, but was ten times lower than that in L. rhamnosus. This result was consistent with the metabolic flux analysis, which indicated that the conversion efficiency of the expressed PFK and FBA was somewhat low. Less than 20 % of the carbons accumulated in the form of fructose-6-phosphate were converted into glyceraldehyde-3-phosphate (GAP) by the expressed PFK and FBA. Metabolic flux analysis also indicated that the enzyme phosphoketolase (XPK) played an important role in splitting the carbon flow from the pentose phosphate pathway to the phosphoketolase pathway. This study suggested that the lactic acid yield of L. brevis could be improved by constructing a homo-fermentative pathway.     

Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet: III. Diurnal Changes in Carbon Partitioning and Carbon Export

The effect of low phosphate supply (low P) was determined on the diurnal changes in the rate of carbon export, and on the contents of starch, sucrose, glucose, and fructose 2,6-bisphosphate (F2,6BP) in leaves. Low-P effects on the activities of a number of enzymes involved in starch and sucrose metabolism were also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers and the low-P treatment induced nutritionally. Low-P treatment decreased carbon export from the leaf much more than it decreased photosynthesis. At growth chamber photon flux density, low P decreased carbon export by 34% in light; in darkness, export rates fell but more so in the control so that the average rate in darkness was higher in low-P leaves. Low P increased starch, sucrose, and glucose contents per leaf area, and decreased F2, 6BP. The total extractable activities of enzymes involved in starch and sucrose synthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5-diphosphoglucose pyrophosphorylase, and sucrose-phosphate synthase. The activities of some enzymes involved in starch and sucrose breakdown were also increased by low P. We propose that plants adapt to low-P environments by increasing the total activities of several phosphatases and by increasing the concentrations of phosphate-free carbon compounds at the expense of sugar phosphates, thereby conserving Pi. The partitioning of carbon among the various carbon pools in low-P adapted leaves appears to be determined in part by the relative capacities of the enzymes for starch and sucrose metabolism.     

Prime Ricerche sulla Fisionomia Enzimatica Caratteristica della Fotosintesi

Summary

The rate of oxidation of glucose-6-phosphate, ribose-5-phosphate, fructose-1,6- phosphate, iso-citrate and malate in extracts from green and etiolated pea leaves was determined, using the triphenyltetrazolium technique.

Glucose-6-phosphate, ribose-5-phosphate, and fructose-1,6-phosphate, in the presence of added TPN, where oxidized at a rate about twice higher in the extracts from green than in the extracts from etiolated leaves. Iso-citrate, in the presence of TPN, fructose-1,6-phosphate, in the presence of DPN, and malate, in the presence of either TPN or DPN, were oxidized at about the same rate in the two types of extracts.

These data seem to indicate a preferential synthesis of enzymes involved in the metabolic cycle of phosphorylated sugars during the transition of the leaf from the etiolated to the photosynthetising physiognomy. They seem also favourable to the view assigning to this metabolic system a primary importance in the anabolic pathway of photosynthesis.     

Rre37 stimulates accumulation of 2-oxoglutarate and glycogen under nitrogen starvation in Synechocystis sp. PCC 6803

Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.     

Relationship between Photosynthetic Electron Transport and Stromal Enzyme Activity in Pea Leaves : Toward an Understanding of the Nature of Photosynthetic Control

The responses of the quantum efficiencies of photosystem (PS) II and PSI measured in vivo simultaneously with estimations of the activities and activation states of NADP-malate dehydrogenase, chloroplast fructose-1,6-bisphosphatase, and ribulose-1,5-bisphosphate carboxylase were used to study the relationship between electron transport and carbon metabolism. The effects of varying irradiance and CO₂ partial pressure on the relationship between the quantum efficiencies of PSI and II, and the activity of these enzymes shows that the interrelationships vary according to the limitations placed on the system. The relationship between the quantum efficiencies of PSII and PSI was linear in most situations. In response to increasing irradiance, the activity of all three enzymes increased. In the case of NADP-malate dehydrogenase this increase was well correlated with the estimated flux of electrons through PSI and PSII. The other two enzymes showed a more complex relationship with the estimated flux of electrons through both photosystems. These relationships are consistent with the known interactions between these stromal enzymes and the thylakoids. The response to varying CO₂ partial pressure is more complex. The efficiencies of PSI and II declined with decreasing CO₂ partial pressure and the activity of each enzyme varied uniquely. However, there are clear correlations between the activities of the enzymes and the flux of electrons through the photosystems. In contrast to the data obtained under conditions of varying irradiance, there is clear evidence of photosynthetic control of electron transport when the CO₂ concentration is varied.     

The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K_m (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

The assimilation of NH₄⁺ causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH₄⁺ assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.     

Light/Dark modulation of enzyme activity in developing barley leaves

Light/dark modulation of the ribulose-5-phosphate kinase, NADP⁺-glyceraldehyde-3-phosphate dehydrogenase, and fructose- 1,6-bisphosphatase activity was measured in the developing primary leaf of barley (Hordeum vulgare L.) seedlings. Ribulose-5-phosphate kinase and NADP⁺ -glyceraldehyde-3-phosphate dehydrogenase were fully light activated even at the earliest developmental stage sampled. In contrast, light modulation of fructose- 1,6-bisphosphatase exhibited a complex response to leaf developmental status. Light stimulation of fructose- 1,6-bisphosphatase activity (measured at pH 8.0) increased progressively during leaf development. On the other hand, acid fructose- 1,6-bisphosphatase activity (measured at pH 6.0) was inhibited by light, and this light inhibition was greater in the base of the leaf than in the tip of the leaf.     

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.     

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.