ADP-glucose Synthesis Research Articles

Regulation of ADPGlucose Synthesis in Guard Cells of Commelina communis

The activator specificity of the ADPglucose pyrophosphorylase from Commelina communis guard cells is the same as observed for the mesophyll cell enzyme. 3-Phosphoglycerate was found to be the most effective activator. Fifty per cent of maximal stimulation was observed at about 100 micromolar. Inorganic phosphate was found to be a potent inhibitor giving 50% inhibition at 0.3 millimolar. These results are discussed with respect to regulation of starch synthesis in guard cells.

Pyrophosphorylases in Solanum tuberosum

ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C(3)-NH(2)). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg(2+) are reported. The enzyme V(max) is stimulated about 1.5- to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed.

Salt Tolerance and Regulation of Enzymes of Starch Synthesis in Cassava (Manihot esculenta Crantz)

The growth rate of cassava plants (Manihot esculenta cv. MAUS7) decreased with increasing concentrations of NaCl from 0 to 75 mM in nutrient solutions supplied regularly in a porous growth medium in a glasshouse. Tuber weight was reduced to one-half between 30 and 50 mM NaCl and there was some burning of apical leaves at 50 and 75 mM NaCl. By comparison with other plants, this cultivar of cassava can be considered to have medium sensitivity to salinity. Na+ and Cl- concentrations increased in all tissues with increasing concentrations of supplied NaCl, except that Na+ remained low in laminae until the 75 mM treatment. K+ levels decreased in tubers. Starch concentrations remained the same in tubers, and K+ stimulated starch bound ADPglucose starch synthase by 1 .5-fold. Leaves and tubers contained activities of ADPglucose pyrophosphorylase and ADPglucose-starch synthase similar to those found in leaves and storage organs of other starch synthesizing plants. ADPglucose pyrophosphorylase from leaves was stimulated 20-fold by 3-phosphoglyceric acid (3PGA) while the enzyme from tubers was almost completely dependent on 3PGA at pH 8.5. The A0.5 values for 3PGA (the concentration required for one-half maximal activation) for the leaf and tuber enzymes at pH 8.5 were 1.31 mM and 7.41 mM respectively. At pH 7.5 the leaf enzyme was stimulated 26-fold and the tuber enzyme was again almost completely dependent on 3PGA. The A0.5 values at pH 7.5 were 1.17 mM and 3.8 mM, respectively. The I0.5 values for PI (concentrations required to cause 50% inhibition) in the presence of 3PGA were 2 mM, 0.25 mM and 0.04 mM for leaf enzyme at pH 8.5 and tuber enzyme at pH 8.5 and 7.7 respectively. The results support the view that it is not possible to generalize about the magnitude of the control of ADPglucose synthesis in leaves as opposed to non-chlorophyllous tissues.

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa.

The ADPglucose pyrophosphorylases from Rhodopseudomonas sphaeroides and Rhodopseudomonas gelatinosa are activated by fructose-6-phosphate, pyruvate and fructose-1,6 biophosphate-P2. The effects of the activators are to increase significantly the Vmax of ADPglucose synthesis and to lower the S0.5 values (concentration of substrates giving 50% maximal velocity) for ATP and MgCl2. The R. sphaeroides enzyme is inhibited by Pi while the R. gelatinosa enzyme is inhibited by AMP as well as by Pi. The interaction between inhibitor and activator is complex. At very low concentrations of activator the enzyme is more sensitized to inhibition. However, at higher concentrations of activator there is a decrease in the sensitivity of the enzyme towards inhibition. The findings are discussed with respect to glycogen synthesis in these microorganisms and may be related to findings that indicate that Rhodopseudomonads have the ability to degrade sugars via the Entner-Duodoroff or Embden-Meyerhoff pathways.

Kinetic properties of Serratia marcescens adenosine 5'-diphosphate glucose pyrophosphorylase.

The regulatory properties of partially purified adenosine 5'-diphosphate-(ADP) glucose pyrophosphorylase from two Serratia marcescens strains (ATCC 274 and ATCC 15365) have been studied. Slight or negligible activation by fructose-P2, pyridoxal-phosphate, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) was observed. These compounds were previously shown to be potent activators of the ADPglucose pyrophosphorylases from the enterics, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Escherichia aurescens, Shigella dysenteriae, and Escherichia coli. Phosphoenolpyruvate stimulated the rate of ADPglucose synthesis catalyzed by Serratia ADPglucose pyrophosphorylase about 1.5- to 2-fold but did not affect the S0.5 values (concentration of substrate required for 50% maximal stimulation) of the substrates, alpha-glucose-1-phosphate, and adenosine 5'-triphosphate. Adenosine 5'-monophosphate (AMP), a potent inhibitor of the enteric ADPglucose pyrophosphorylase, is an effective inhibitor of the S. marcescens enzyme. ADP also inhibits but is not as effective as AMP. Activators of the enteric enzyme counteract the inhibition caused by AMP. This is in contrast to what is observed for the S. marcescens enzyme. Neither phosphoenolpyruvate, fructose-diphosphate, pyridoxal-phosphate, NADPH, 3-phosphoglycerate, fructose-6-phosphate, nor pyruvate effect the inhibition caused by AMP. The properties of the S. marcescens HY strain and Serratia liquefaciens ADPglucose pyrophosphorylase were found to be similar to the above two S. marcescens enzymes with respect to activation and inhibition. These observations provide another example where the properties of an enzyme found in the genus Serratia have been found to be different from the properties of the same enzyme present in the enteric genera Escherichia, Salmonella, Shigella, Citrobacter, and Enterobacter.

Biosynthesis of bacterial glycogen IX: Regulatory properties of the adenosine diphosphate glucose pyrophosphorylases of the Enterobocteriaceae

The ADP-glucose pyrophosphorylases from several enteric organisms were found to be activated by fructose diphosphate, NADPH, pyridoxal-5′-P, and by some other glycolytic intermediates. The enzyme was partially purified from extracts of Salmonella typhimurium and Citrobacter freundii and the effects of the activators on the kinetic properties of the enzymes were studied. The activators increased the apparent affinities of the enzyme for the various substrates and increased the maximal velocity of ADP-glucose synthesis several-fold for both partially purified enzymes. Both enzymes are inhibited by AMP, ADP, and P i with AMP being the most potent inhibitor. The concentration of activator modulated the sensitivity of the S. typhimurium enzyme towards inhibition by AMP, ADP, or P i. The enzyme is more sensitive to inhibition at lower and unsaturating concentration of the activators. However, in the absence of activator only 50–80% of the ADP-glucose synthesis rate could be inhibited even at very high concentrations of 5′AMP (∼2.5 m m) while in the presence of activator greater than 95% inhibition of ADP-glucose synthesis occurred at concentrations of 0.5 m m AMP or less. The above findings are discussed with respect to the regulation of glycogen synthesis in the Enterobacteriaceae.

Biosynthesis of bacterial glycogen: VIII. Activation and inhibition of the adenosine diphosphoglucose pyrophosphorylases of Rhodopseudomonas capsulata and of Agrobacterium tumefaciens

The ADP-glucose pyrophosphorylases from R. capsulata and A. tumefaciens are found to be activated by fructose-6-P and to lesser extents by pyruvate, KDPG, ribose-5-P, and deoxyribose-5-P. Fructose-6-P increases the V max of synthesis of ADP-glucose for both enzymes about 6-fold. In addition the S 0.5 values (concentrations of substrates giving 50% of maximal velocity) for ATP and glucose-1-P for both enzymes are decreased in the presence of the activator, fructose-6-P. Both enzymes are inhibited by P i, AMP, ADP, and phosphoenolpyruvate. However the presence of fructose-6-P decreases the sensitivities of the enzymes to inhibition by the above compounds. The concentration of P i, AMP, ADP, and PEP required for 50% inhibition in the absence of fructose-6-P are 0.26, 0.64, 0.41, and 0.047 m m, respectively. These values are increased to 5.8, 7.3, 3.8, and 1.6 m m, respectively, in the presence of 1.0 m m fructose-6-P. These findings are discussed with respect to glycogen synthesis in these microorganisms and are related to the findings that indicate that both R. capsulata and A. tumefaciens catabolize glucose mainly via the Entner-Doudoroff pathway.

TPNH and pyridoxal-5′-phosphate: Activators of ADP-glucose pyrophosphorylase of Escherichia coli B

TPNH and pyridoxal-5-P have been found to be potent activators of E. coli B ADP-glucose pyrophosphorylase. The concentrations required for 50% maximal activation was 0.15 mM TPNH and 8 μM pyridoxal-5-P. These values compare favorably to the value of 0.13 mM found previously for the activator, fructose diP. Kinetic evidence is presented to suggest all three activators bind to the same site(s) on the enzyme. PLP-activated ADP-glucose synthesis is less sensitive to 5′-adenylate inhibition than is TPNH-activated synthesis. The physiological functions of these activations with respect to glycogen storage is discussed.

Biosynthesis of Bacterial Glycogen: VII. Purification and Properties of the Adenosine Diphosphoglucose Pyrophosphorylase of Rhodospirillum Rubrum

Abstract The adenosine diphosphate glucose pyrophosphorylase from Rhodospirillum rubrum has been purified to apparent homogeneity with a heat step, protamine sulfate fractionation, ammonium sulfate fractionation, DEAE-Sephadex chromatography, and two preparative disc gel electrophoresis steps. The molecular weight obtained from this enzyme from sedimentation equilibrium was from 195,000 (for v = 0.70) to 245,000 (for v = 0.75). A molecular weight of 225,000 was obtained by a disc gel electrophoresis technique. The synthesis of ADP-glucose was dependent on the presence of ATP, glucose-1-P, enzyme, and either magnesium or manganous ion. The enzyme was quite specific for ATP. CTP, dGTP, ITP, TTP, and UTP could not substitute for ATP. In the presence of pyruvate, CTP gave 4.4% and dATP 1.9% of the activity found when ATP was the substrate. The enzyme was activated by pyruvate. None of the other glycolytic intermediates tested activated this enzyme. Pyruvate (5 mm) increased the Vmax of ADPglucose synthesis 2.5- to 3-fold, decreased the S0.5 for ATP about 10-fold (from 3.4 mm to 0.36 mm), and shifted the pH optimum of the enzyme from pH 8.5 to a broader pH optimum extending from pH 7.5 to 9.2. The saturation curves for ATP and pyruvate were sigmoidal, and a reciprocal heterotropic interaction exists between these two ligands. Increasing the concentration of one of these ligands decreased the sigmoidicity of the saturation curve for the other, as well as the concentration needed for half-maximal activity. The Hill constant (n) for pyruvate was 1.9 at 0.25 mm ATP, 1.7 at 0.50 mm ATP, and 1.2 at 2.5 mm ATP. n for ATP was 2.97 in the absence of pyruvate and 1.82 in the presence of 5 mm pyruvate. It was also found that pyruvate decreased the amount of metal ion required for half-maximal velocity: from 6.0 mm to 2.7 mm with Mg++, and from 5.4 mm to 1.8 mm with Mn++. When the level of ATP was below 1 mm, and Mg++ below 2 mm, the enzyme was almost totally dependent on pyruvate for activity. At 0.5 mm ATP, pyruvate in the range of concentration of 0.1 mm to 3 mm dramatically increased the activity of the enzyme. The reverse reaction, the pyrophosphorolysis of ADP-glucose, was dependent on the presence of ADP-glucose, enzyme, and Mg++. Pyruvate increased the Vmax of ADP-glucose pyrophosphorolysis from 1.5- to 2-fold, decreased the S0.5 for ADP-glucose more than 5-fold (from 2.0 mm to 0.38 mm), and decreased the Hill constant n with respect to ADP-glucose from n = 2.02 to n = 1.42.

Regulation of starch biosynthesis in plant leaves: activation and inhibition of ADPglucose pyrophosphorylase.

The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 65 degrees for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate. Fructose-6-phosphate and fructose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents. The A(0.5) (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 x 10(-6)m while for the sorghum enzyme it was 3.7 x 10(-4)m. Inorganic phosphate proved to be an effective inhibitor of ADPglucose synthesis. The I(0.5) (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 x 10(-5)m (barley) to 1.9 x 10(-4)m (sorghum). This inhibition was reversed or antagonized by the activator 3-phosphoglycerate. These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis.

Biosynthesis of starch in chloroplasts.

The enzymic synthesis of ADP-glucose and UDP-glucose by chloroplastic pyrophosphorylase of bean and rice leaves has been demonstrated by paper chromatographic techniques. In both tissues, the activity of UDP-glucose-pyrophosphorylase was much higher than ADP-glucose-pyrophosphorylase. Glycerate-3-phosphate, phosphoenolpyruvate and fructose-1,6-diphosphate did not stimulate ADP-glucose formation by a pyrophosphorylation reaction. The major metabolic pathway for UDP-glucose utilization appears to be the synthesis of either sucrose or sucrose-P. On the other hand, a specific precursor role of ADP-glucose for synthesizing chloroplast starch by the ADP-glucose-starch transglucosylase reaction is supported by the coupled enzyme system of ADP-glucose-pyrophosphorylase and transglucosylase, isolated from chloroplasts. None of the glycolytic intermediates stimulated the glucose transfer in the enzyme sequence of reaction system employed.

Biosynthesis of starch in Chlorella pyrenoidosa: II. Regulation of ATP: α- d-Glucose 1-phosphate adenyl transferase (ADP-Glucose pyrophosphorylase) by inorganic phosphate and 3-phosphoglycerate

ADP-Glucose pyrophosphorylase from Chlorella pyrenoidosa has been partially purified and its properties have been investigated. The enzyme activity was activated about 20-fold by 3-phosphoglyceric acid, the first product of the photosynthetic reduction cycle. The enzyme appears to be separate and distinct from UDP-glucose pyrophosphorylase, which was not activated by 3-phosphoglycerate. ADP-Glucose and ATP gave sigmoid-shaped rate versus concentration curves in the presence or absence of 3-phosphoglycerate. The V max of pyrophosphorolysis and synthesis of ADP-glucose was increased 7-fold and 18-fold respectively by 3-phosphoglycerate, and the K values of ADP-glucose and ATP were decreased 1.6-fold in the presence of the activator. Inorganic phosphate and ADP were inhibitors of the enzyme. The K i for phosphate and ADP were 2 × 10 −4 m and 2.5 × 10 −3 m, respectively. The inhibition by phosphate can be reversed by high concentrations of the activator, 3-phosphoglycerate. However, only partial reversal of ADP inhibition occurred with high concentrations of 3-phosphoglycerate. The significance of 3-phosphoglycerate activation and inhibition by phosphate and ADP in the regulation of starch biosynthesis in Chlorella was discussed.

Biosynthesis of bacterial glycogen: V. The activation and inhibition of the adenosine diphosphate glucose pyrophosphorylase of Arthrobacter viscosus NRRL B1973

Adenosine diphosphate-glucose pyrophosphorylase from Arthrobacter viscosus NRRL B1973 was found to be activated by fructose-6-P, pyruvate, deoxyribose-5-P and ribose-5-P. Fructose-6-P was the most effective activator. Fructose-6-P increased the V max of pyrophosphorolysis of ADP-glucose 2.5-fold, and the V max of synthesis of ADP-glucose from ATP and glucose-1-P, 7-fold. The apparent K m 's for ATP, pyrophosphate, and ADP-glucose were decreased in the presence of fructose-6-P. Pyruvate increased the V max of pyrophosphorolysis 2-fold and of synthesis 6-fold. However, pyruvate only decreased the K m for pyrophosphate. The K m for glucose-1-P was not effected by either pyruvate or fructose-6-P. The enzyme was found to be inhibited by phosphate, sulfate, AMP, ADP, phosphoenolpyruvate, GMP, and GDP. However, fructose-6-P completely overcame (reversed) these inhibitions. The relationship of these findings to the control of bacterial glycogen synthesis is discussed.