Ultrasensitive glycogen synthesis in Cyanobacteria. (1/172)

Cyanobacter ADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3-phosphoglycerate, elicited by orthophosphate and polyethyleneglycol-induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15-19-fold with respect to 1% of the maximal system velocity. Only a 2.4-3.8-fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200-fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.  (+info)

A phosphoglycerate to inorganic phosphate ratio is the major factor in controlling starch levels in chloroplasts via ADP-glucose pyrophosphorylase regulation. (2/172)

Purified barley leaf ADP-glucose pyrophosphorylase, a key enzyme of the starch synthesis in the chloroplast stroma, was analysed with respect to its possible regulation by factors defining the metabolic/effector status of the chloroplast during light and dark conditions. The enzyme required 3-phosphoglyceric acid for the maximal activity and was inhibited by inorganic phosphate. The optimal pH for the enzyme was at circa 7.0, regardless of the presence or absence of 3-phosphoglyceric acid, whereas the maximal activation by 3-phosphoglyceric acid was observed at pH 8.5 and higher. Changes in the concentration of Mg2+ and dithiothreitol had little or no effect on the enzymatic activity of AGPase. It has been directly demonstrated for the first time that a 3-phosphoglyceric acid/inorganic phosphate ratio, a crucial regulatory parameter, could be directly related to a defined activation state of the enzyme, allowing the prediction of a relative AGPase activity under given conditions. The predicted changes in the enzyme activity were directly correlated with earlier reported responses of starch levels to the 3-phosphoglyceric acid/inorganic phosphate ratio in chloroplasts. Consequences of this for the starch biosynthesis are discussed.  (+info)

Auxin and cytokinin have opposite effects on amyloplast development and the expression of starch synthesis genes in cultured bright yellow-2 tobacco cells. (3/172)

In cultured Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells, the depletion of auxin (2,4-dichlorophenoxyacetic acid) in the culture medium induces the accumulation of starch. This is accelerated by the addition of cytokinin (benzyladenine). Light and electron microscopic observations revealed that this amyloplast formation involves drastic changes in plastid morphology. The effects of auxin and cytokinin on amyloplast development were investigated by adding auxin or cytokinin to cells grown in a hormone-free culture. Auxin repressed amyloplast development, whereas cytokinin accelerated starch accumulation regardless of the timing of hormone addition. RNA gel-blot analysis revealed that the accumulation of the ADP-glucose pyrophosphorylase small subunit gene (AgpS), granule-bound starch synthase, and starch branching enzyme transcripts were also affected by hormonal conditions. High levels of AgpS, granule-bound starch synthase, and starch branching enzyme transcripts accumulated in amyloplast-developing cells grown in auxin-depleted conditions. Furthermore, the addition of auxin to the cells cultured in hormone-free medium reduced the level of AgpS transcripts, whereas the addition of cytokinin increased it, irrespective of the timing of hormone addition. These results suggest that auxin and cytokinin exert opposite effects on amyloplast development by regulating the expression of the genes required for starch biosynthesis.  (+info)

Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis. (4/172)

Sucrose synthase (Sus) is a key enzyme of sucrose metabolism. Two Sus-encoding genes (Sus1 and Sus2) from Arabidopsis thaliana were found to be profoundly and differentially regulated in leaves exposed to environmental stresses (cold stress, drought or O(2) deficiency). Transcript levels of Sus1 increased on exposure to cold and drought, whereas Sus2 mRNA was induced specifically by O(2) deficiency. Both cold and drought exposures induced the accumulation of soluble sugars and caused a decrease in leaf osmotic potential, whereas O(2) deficiency was characterized by a nearly complete depletion in sugars. Feeding abscisic acid (ABA) to detached leaves or subjecting Arabidopsis ABA-deficient mutants to cold stress conditions had no effect on the expression profiles of Sus1 or Sus2, whereas feeding metabolizable sugars (sucrose or glucose) or non-metabolizable osmotica [poly(ethylene glycol), sorbitol or mannitol] mimicked the effects of osmotic stress on Sus1 expression in detached leaves. By using various sucrose/mannitol solutions, we demonstrated that Sus1 was up-regulated by a decrease in leaf osmotic potential rather than an increase in sucrose concentration itself. We suggest that Sus1 expression is regulated via an ABA-independent signal transduction pathway that is related to the perception of a decrease in leaf osmotic potential during stresses. In contrast, the expression of Sus2 was independent of sugar/osmoticum effects, suggesting the involvement of a signal transduction mechanism distinct from that regulating Sus1 expression. The differential stress-responsive regulation of Sus genes in leaves might represent part of a general cellular response to the allocation of carbohydrates during acclimation processes.  (+info)

Is leaf ADP-glucose pyrophosphorylase an allosteric enzyme? (5/172)

Barley leaf ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch synthesis in the chloroplast stroma, was analysed, in both directions of the reaction, with respect to details of its regulation by 3-phosphoglycerate (PGA) and inorganic phosphate (Pi) which serve as activator and inhibitor, respectively. AGPase was found to catalyse a close-to-equilibrium reaction, with the K(eq) value of approximately 0.5, i.e. slightly favouring the pyrophosphorolytic direction. When the enzyme was analysed by substrate kinetics, PGA acted either as a linear (hyperbolic response) 'non-competitive' activator (forward reaction) or a linear near-'competitive' activator (reverse reaction). When the activation and inhibition patterns with PGA and Pi, respectively, were studied in detail by Dixon plots, the response curves to effectors also followed hyperbolic kinetics, with the experimentally determined K(a) and K(i) values on the order of micromolar. The results suggest that the regulation of AGPase proceeds via a non-cooperative mechanism, where neither of the effectors, when considered separately, induces any allosteric response. The evidence, discussed in terms of an overall kinetic mechanism/regulation of leaf AGPase, prompts caution in classifying the protein as an 'allosteric enzyme'.  (+info)

Activation of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin. (6/172)

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, P(i). It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys(12) disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 microM) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 microM for reduced thioredoxin f and m, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys(12) is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch.  (+info)

Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture. (7/172)

During the growth of Clostridium cellulolyticum in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in C. cellulolyticum was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262-3269, 1999).  (+info)

Isolation and characterization of polymorphic cDNAs partially encoding ADP-glucose pyrophosphorylase (AGPase) large subunit from sweet potato. (8/172)

cDNA clones encoding sweet potato AGPase large subunit (iAGPLI) from the cDNA library constructed from the tuberous root were isolated. Two clones were characterized and named iAGPLI-a and iAGPLI-b. They were 1,661 bp and 1,277 bp in length and contained partial open reading frames of 450 and 306 amino acids, respectively. Both nucleic acid and amino acid sequence identities between iAGPLI-a and iAGPLI-b were 83.8% and 97.3%, respectively. Based on the amino acid sequence analysis, iAGPLI-a and iAGPLI-b share the highest sequence identity (81%) with potato AGPase large subunit. The iAGPLI-a and iAGPLI-b genes were expressed predominantly in the stem and weakly in the tuberous root, and no transcript was expressed in other tissues. The sweet potato genome contains several copies of the iAGPLI gene.  (+info)