A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG)

The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step ¹⁴C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- α -arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.     

beta-Maltose is the metabolically active anomer of maltose during transitory starch degradation

Maltose is the major form of carbon exported from the chloroplast at night as a result of transitory starch breakdown. Maltose exists as an alpha- or beta-anomer. We developed an enzymatic technique for distinguishing between the two anomers of maltose and tested the accuracy and specificity of this technique using beta-maltose liberated from maltoheptose by beta-amylase. This technique was used to investigate which form of maltose is present during transitory starch degradation in bean (Phaseolus vulgaris), wild-type Arabidopsis (Arabidopsis thaliana), two starch deficient Arabidopsis lines, and one starch-excess mutant of Arabidopsis. In Phaseolus and wild-type Arabidopsis, beta-maltose levels were low during the day but were much higher at night. In Arabidopsis plants unable to metabolize maltose due to a T-DNA insertion in the gene for the cytosolic amylomaltase, (Y. Lu, T.D. Sharkey [2004] Planta 218: 466-473) levels of alpha- and beta-maltose were high during both the day and night. In starchless mutants of Arabidopsis, total maltose levels were low and almost completely in the alpha-form. We also found that the subcellular concentration of beta-maltose at night was greater in the chloroplast than in the cytosol by 278 microm. We conclude that beta-maltose is the metabolically active anomer of maltose and that a sufficient gradient of beta-maltose exists between the chloroplast and cytosol to allow for passive transport of maltose out of chloroplasts at night.     

Lytic bodies from cereals hydrolysing maltose and starch

Protein bodies and spherosomes from sorghum contained carbohydrase activity against maltose, starch and p-nitrophenyl-α-d-glucoside. Maltase activities in sorghum and also in maize lytic bodies were very high; carbohydrase activities of lytic bodies from whole wheat, whole barley, sorghum aleurone, sorghum embryo and maize embryo were considerably lower. The pH response of sorghum lytic bodies was bimodal with an optimum in the range of 3·4–4·2 and a minimum or a shoulder near pH 3·8. Protein bodies from sorghum, maize, wheat and barley reduced the iodine-colouring capacity of soluble starch to give a purple colour typical of a β-limit dextrin. With spherosomes colour reduction was usually more rapid, eventually taking the breakdown of starch beyond the achroic point. The lytic bodies produce both maltose and glucose from starch, except in the case of maize when only glucose was found. The data suggest that protein bodies contain a linked β-amylase-maltase system and that spherosomes contain a linked α-amylase-maltase system.     

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.     

Fermentation of maltose and starch by Klebsiella oxytoca P2

Klebsiella oxytoca P2, which has genes from Zymomonas mobilis encoding the alcohol dehydrogenase and pyruvate decarboxylase integrated in its chromosome, fermented 50 g maltose/l to 25.4 g of ethanol/l. It also fermented 10, 20 and 40 g starch/l yielding 4, 8.4, and 17.7 g ethanol/l, respectively, representing 72, 75 and 78% of the theoretical yield. © Rapid Science Ltd. 1998     

Oxidation and degradation of maltose and isomaltose by persulfate

Maltose and isomaltose were oxidized and degraded by ammonium persulfate in 66.7 mmol aqueous phosphate buffer (pH 7.1). Product and e.s.r. studies suggested that radicals formed by C-H abstraction at C-1 and C-4 were predominant for oxidation of maltose, whereas C-H abstraction of isomaltose proceeded preferentially at C-5 of the reducing glucose group.     

Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

Mutants of Arabidopsis lacking starch branching enzyme II substitute plastidial starch synthesis by cytoplasmic maltose accumulation

Three genes, BE1, BE2, and BE3, which potentially encode isoforms of starch branching enzymes, have been found in the genome of Arabidopsis thaliana. Although no impact on starch structure was observed in null be1 mutants, modifications in amylopectin structure analogous to those of other branching enzyme II mutants were detected in be2 and be3. No impact on starch content was found in any of the single mutant lines. Moreover, three double mutant combinations were produced (be1 be2, be1 be3, and be2 be3), and the impact of the mutations on starch content and structure was analyzed. Our results suggest that BE1 has no apparent function for the synthesis of starch in the leaves, as both be1 be2 and be1 be3 double mutants display the same phenotype as be2 and be3 separately. However, starch synthesis was abolished in be2 be3, while high levels of alpha-maltose were assayed in the cytosol. This result indicates that the functions of both BE2 and BE3, which belong to class II starch branching enzymes, are largely redundant in Arabidopsis. Moreover, we demonstrate that maltose accumulation depends on the presence of an active ADP-glucose pyrophosphorylase and that the cytosolic transglucosidase DISPROPORTIONATING ENZYME2, required for maltose metabolization, is specific for beta-maltose.     

Temperature effects on circadian clocks

Periodic temperature changes represent one of the most effective entraining (Zeitgeber) signals for circadian clocks in many organisms. Different constant temperatures affect the circadian amplitude and ultimately the expression of circadian clocks, while the circadian period length (tau) remains approximately constant (temperature compensation). Experimental results and theoretical models are presented that may serve to explain these effects. After introducing the physico-chemical basis of temperature on enzyme-catalyzed and physiological reactions, and after describing mechanisms for temperature adaptation of physiological reactions to different thermal environments, general effects of temperature on chemical and biological oscillators are described. Kinetic models for circadian clocks and temperature compensation are presented and compared with experimental results. Special attention is given to the question how constant but different temperature levels affect clock amplitude, period length and phase. Influences of single and periodic temperature variations (steps or pulses) on circadian clocks are presented together with models which may explain the resulting phase response curves and entrainment patterns. Because temperature compensation is only one aspect of a general homeostatic mechanism that keeps the circadian period rather constant, the influence of other environmental variables and their relationship to temperature are discussed.     

Crosstalk between xenobiotics metabolism and circadian clock

Many aspects of physiology and behavior in organisms from bacteria to man are subjected to circadian regulation. Indeed, the major function of the circadian clock consists in the adaptation of physiology to daily environmental change and the accompanying stresses such as exposition to UV-light and food-contained toxic compounds. In this way, most aspects of xenobiotic detoxification are subjected to circadian regulation. These phenomena are now considered as the molecular basis for the time-dependence of drug toxicities and efficacy. However, there is now evidences that these toxic compounds can, in turn, regulate circadian gene expression and thus influence circadian rhythms. As food seems to be the major regulator of peripheral clock, the possibility that food-contained toxic compounds participate in the entrainment of the clock will be discussed.     

Solvent effects on starch dissolution and gelatinization

The disruption of starch granular structure during dissolution in varying concentrations of N-methyl morpholine N-oxide (NMMO) has been studied using three maize starches with varying ratios of amylose and amylopectin. Behavior in NMMO has been characterized by differential scanning calorimetry (DSC), microscopy, rapid viscosity analysis (RVA), and rheometry. Exothermic transitions were observed for the three starches in both 78 and 70% NMMO; the transition changed to an endotherm at 60 and 50% NMMO. Consistent with DSC, hot stage microscopy showed that starch granules dissolved at NMMO concentrations of 78 and 70%, whereas in 60 and 50% NMMO, gelatinization behavior similar to that found for starch in water was observed. Mechanical spectroscopy revealed the dominant viscous behavior (G″ > G') of starch at NMMO concentrations of 70 and 78% and more elastic behavior (G' > G″) at lower concentrations. Starch solutions in 78% NMMO obey the Cox-Merz rule, suggesting that the solutions are homogeneous on a molecular level.     

The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.     

Kinetics of condensation of glucose into maltose and isomaltose in hydrolysis of starch by glucoamylase

Kinetics of the condensation of glucose into maltose and isomaltose in the hydrolysis of starch by two types of glucoamylase (from Aspergillus niger and Rhizopus niveus) was studied both experimentally and theoretically. A kinetic model for the hydrolysis of starch by glucoamylase from A. niger was proposed. In this model the reversible hydrolysis of maltose and isomaltose and the kinetic parameters change were taken into consideration. Calculated values agreed approximately with the experimental results, and this simple kinetic model was found to have practical use. The rate of condensation of glucose into isomaltose by enzyme from A. niger was about three times larger than that by enzyme from R. niveus. At a higher initial concentration of starch a large amount of isomaltose was reversed, and the glucose yield was reduced significantly after very long reaction times.