Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max

The aim of this work was to determine the relative contributions of ADPglucose and UDPglucose to starch synthesis in two non-photosynthetic tissues, the developing club of the spadix of Arum maculatum and suspension cultures of Glycine max. Rates of starch accumulation during growth are compared with estimates of the maximum catalytic activities in vitro of ADPglucose starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and UDPglucose starch synthase. The latter could only be measured at high concentrations (10–30 mM) of UDPglucose. Clubs of Arum and cells of Glycine contained 292 and 6.8 nmol UDPglucose per gram fresh weight, respectively. The corresponding figures for ADPglucose were 29 and 0.4. From the above data it is argued that in both Arum club and Glycine cells the activity of UDPglucose starch synthase is too low to make any quantitatively significant contribution to starch synthesis. The activities of ADPglucose starch synthase and pyrophosphorylase were high enough to mediate the observed rates of starch accumulation. It is suggested that starch synthesis in these tissues is via ADPglucose.     

Escherichia coli K-12 glycogen synthase: ability to use UDPglucose and ADP glucose as glucosyl donors in the absence of added primer.

A mechanism of initiation of glycogen biosynthesis in Escherichia coli has been previously postulated: In a first step, the glucosyl groups would be transferred into an acceptor protein from UDPglucose or ADPglucose by two glucosyl transferases, distinct from the glycogen synthase. In this work, the activity of transfer from UDPglucose into a methanol-insoluble fraction could not be found in the crude extracts of six independently isolated glycogen synthase-deficient mutants of E. coli K-12. Purified E. coli K-12 glycogen synthase was able to catalyze the unprimed reaction from ADPglucose and UDPglucose but at a very low rate; the rate with UDPglucose is 6–7% the rate observed with ADPglucose. With these two substrates, the unprimed reaction was strongly stimulated by the simultaneous presence of salts and branching enzyme. However the activity with UDPglucose increased rapidly at low concentrations of branching enzyme and was inhibited at physiological concentrations whereas the activity with ADPglucose reached a maximum only at these concentrations. Consequently, the relative activities found with ADPglucose and UDPglucose varied with the branching enzyme concentration. Transfer from UDPglucose was inhibited by low concentrations of ADPglucose and high concentrations of glycogen. These results suggest that the same enzyme, namely the glycogen synthase, catalyzes the unprimed transfer from ADPglucose and UDPglucose and that ADPglucose is probably the most important physiological donor in glycogen biosynthesis in E. coli.     

Starch Synthesis in Developing Potato Tubers

The activities of enzymes involved in starch metabolism were measured at intervals during tuberization and the early stages of tuber growth in Solanum tubersum grown in water culture under controlled environmental conditions. Starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase activities all increased during tuber development, the most pronounced increases occurring in the activities of ADP-glucose pyrophosphorylase and phosphorylase. The activity ratio ADPglucose pyrophosphorylase/phosphorylase was lowest in slow growing tubers and hightest in fast growing tubers. In addition, high sugar concentrations in fast growing tubers and low sugar concentrations in slow growing tubers suggested that enzyme levels might be influenced by sugar concentration. The activities of starch synthase, phosphorylase and ADPglucose pyrophosphorylase were increased 2–2.5 fold by the presence of 100 mM K⁺. It is concluded that the major enzyme changes occur as a consequence of tuber initiation and that starch accumulation is controlled, at least in part, by the activities of ADPglucose pyrophosphorylase and phosphorylase.     

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.     

Changes in the activity of phosphogluconate dehydrogenase and its regulation in tobacco infected with PVY

The content and changes in the activity of phosphogluconate dehydrogenase were followed in leaf tissues of tobacco plants infected with the potato virus Y (PVY) in the acute phase of infection. The activity of the enzyme was higher in virus-infected tissues during the entire experimental period compared with the values found in healthy control plants in both crude homogenate and partially purified enzyme preparation. The courses of the activity curves of both the crude extract and partially purified enzyme preparations were consistent and correlated with the reproduction curve of the virus. These results suggest that increased activity of the enzyme was a result of its coarse regulation and indicate its immediate involvement in“de novo” biosynthesis of the virus via the oxidative pentose phosphate pathway.     

Biosynthesis of bacterial glycogen: Kinetic studies of a glucose-1-P adenylyltransferase (EC 2.7.7.27) from a glycogen-excess mutant of Escherichia coli B

An Escherichia coli B mutant, CL1136 accumulates glycogen at 3.4 to 4 times the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to α- and β-amylolysis, chain length determination and I₂-complex absorption spectra. The CL1136 mutant contains normal glycogen synthase and branching enzyme activity but has an ADPglucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and in contrast to the present strain enzyme studied previously, is highly active in the absence of the allosteric activator. The response of the CL1136 enzyme to energy charge has been determined and this enzyme shows appreciable activity at low energy charge values where the E. coli B enzyme is inactive. The response to energy charge for the CL1136 and E. coli B enzymes are correlated with the rates of glycogen accumulation observed in the microorganisms. The regulation of glycogen synthesis in E. coli is to a great extent at the level of ADPglucose pyrophosphorylase; varying concentrations of fructose-P₂ and energy charge determine the rate of ADPglucose and glycogen synthesis. Both the allosteric regulation of ADPglucose pyrophosphorylase as well as the genetic regulations of the synthesis of glycogen biosynthetic enzymes (glycogen synthase and ADPglucose pyrophosphorylase) are involved in the regulation of glycogen accumulation in E. coli B.     

Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity

A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F₁ progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

Studies on the effect of temperature on the activity and stability of cyanobacterial ADP-glucose pyrophosphorylase

The effect of temperature on the activity and stability of ADPglucose pyrophosphorylase from Anabaena PCC 7120 was studied. Experimental optima temperatures were found around 37-40 degrees C or 42-45 degrees C, depending on the absence or the presence of allosteric effectors in the assay medium, respectively. In the range of temperature where the enzyme is stable, curved Arrhenius plots were obtained, indicating a transition temperature between 9 and 12 degrees C. Since these results were observed for both the forward and reverse reaction, with two different sets of substrates and two entirely different assay procedures, it seems unlikely that the effect can be on any component of the system other than the enzyme itself. Results suggest that cyanobacterial ADPglucose pyrophosphorylase undergoes conformational changes at different temperatures, rendering structures with different catalytic efficiencies. The different structures of the enzyme were visualized by emission fluorescence. ADPglucose pyrophosphorylase was irreversibly inactivated when exposed to temperatures above 40 degrees C. Inactivation was dependent on temperature and followed first order kinetics. The substrate, ATP, and the allosteric effectors, 3PGA and Pi, effectively protected the enzyme against thermal inactivation. Protection afforded by ATP was affected by MgCl2. These results suggest that the binding of the effectors to the enzyme resulted in conformational changes of the protein, rendering structures more stable to temperature treatments. Similar structures could be adopted by the enzyme in different environments, since the higher stability was observed in media containing either high ionic strength or high hydrophobicity.     

Characterization of Escherichia coli B glycogen synthase enzymatic reactions and products.

Previous reports implicate UDPglucose as an active glucosyl donor for the unprimed reaction and “glucoprotein” formation in glycogen biosynthesis in Escherichia coli. Results presented here indicate that UDPglucose and GDPglucose are glucosyl donors in the primed and unprimed reactions catalyzed by purified E. coli B glycogen synthase at less than 5% the rate observed when ADPglucose is the donor. The unprimed reaction is stimulated by 0.25 m citrate and a high molecular weight product is formed similar to that produced when ADPglucose is the glucosyl donor. Physiological amounts of branching enzyme and high concentrations of glycogen inhibit transfer from UDPglucose and GDPglucose. In addition, transfer from UDPglucose is inhibited by ADPglucose. These results strongly suggest that ADPglucose is the physiological donor in both the primed and unprimed reactions. Furthermore, these and previously reported results suggest that one enzyme is involved in the catalysis of the primed, unprimed, and TCA-insoluble product formation reactions. Antiserum prepared against purified E. coli B glycogen synthase inactivates transfer of glucose from either ADPglucose or UDPglucose in the above reactions catalyzed by E. coli B crude extracts. Purified E. coli B glycogen synthase preparations contain significant amounts of α-glucan primer. Evidence shows that this glucan is not covalently attached to the enzyme. Results presented show that formation of material insoluble in TCA and previously considered to be due to “glucoprotein” formation, is in fact due to the generation of long chain length glucan molecules intrinsically acid insoluble. The data suggest that previous results purported to be de novo synthesis of glycogen are due to glucan associated with the glycogen synthase and not to formation of a “glucoprotein” intermediate which then acts as primer for further oligosaccharide synthesis.     

Regulation of metabolic pathways PVY-RNA biosynthesis in tobacco: Host’s RNA degradation

Tobacco plants infected with the potato virus Y (PVY) were studied during the acute-infection period. The control enzymes of metabolic pathway of hosts RNA degradation tending to biosynthesis of PVY-RNA, its coarse/fine regulation and content of hosts RNA were monitored. Activities of ribonucleases, phosphomonoesterases and phosphodiesterases in both the crude homogenates and the partially purified enzyme preparations from the diseased leaves were markedly increased when compared to the tissues from healthy plants. The curves of enzyme activities positively correlated with the multiplication curve of the PVY and negatively correlated with the decreased contents of hosts RNA. The enzyme activity in homogenate samples did not significantly differ from the corresponding purified enzyme preparations.     

Purification of Spinach Leaf ADPglucose Pyrophosphorylase

ADPglucose pyrophosphorylase from spinach leaves has been purified to homogeneity by hydrophobic chromatography carried out in 1 molar phosphate buffer. After polyacrylamide gel electrophoresis, the preparation showed only one protein staining band that coincided with a single activity stain. The enzyme appears to be composed of two subunits with molecular weights of 44,000 and 48,000, respectively, as determined by SDS polyacrylamide gel electrophoresis. Thus ADPglucose pyrophosphorylase of spinach appears to be comprised of subunits which are similar in size to the subunits of ADPglucose pyrophosphorylase isolated from bacterial sources. In contrast, a subunit molecular weight of 96,000 has been reported for the maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). The purified enzyme retains similar allosteric and catalytic properties as reported previously and is more sensitive to phosphate inhibition under “dark”-simulated conditions than under “light”-simulated conditions.     

Initiation of glycogen biosynthesis in Escherichia coli studies of the properties of the enzymes involved

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied.It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4°C.The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period.The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein.Both UDPglucose and ADPglucose: protein glucosyltransferases were affected in different ways by detergents.Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen.The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.     

Pyrophosphorylases in Solanum tuberosum: III. PURIFICATION, PHYSICAL, AND CATALYTIC PROPERTIES OF ADPGLUCOSE PYROPHOSPHORYLASE IN POTATOES

ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C₃-NH₂). 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 Mg²⁺ 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.     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> HW2 enhanced cucumber resistance against cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of cucumber plants worldwide. In the present study, we use plant growth-promoting rhizobacteria (PGPR) to control this virus effectively. Stenotrophomonas maltophilia HW2 was isolated from healthy cucumber root, exhibited a good biocontrol efficacy against CGMMV. Here, it is documented that 20 d after virus inoculation, the biocontrol efficacy of HW2 reached 52.61%. HW2 can effectively colonize in cucumber rhizosphere, and also promoted cucumber plants growth. We also examined the effect of HW2 on viral replication and its mechanism. Compared with the control, HW2 pre-treated plants could delay virus replication for more than 3 d and inhibit viral protein genes (CP, MP, Rep) expression in the cucumber leaf. The expression of antioxidant enzyme genes (SOD and CAT) and defense-related genes (PR1 and PR5) were quickly induced by HW2. These results suggest that HW2 induced plant defense responses to CGMMV by increasing the expression of defense response genes. We report for the first time that Stenotrophomonas maltophilia improved cucumber resistance against CGMMV, which highlights the applying of PGPR on controlling of virus diseases.     

Initiation of glycogen biosynthesis in Escherichia coli. Studies of the properties of the enzymes involved.

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied. It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4 degrees C. The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period. The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein. Both UDPglucose and ADPglucose : protein glucosyltransferases were affected in different ways by detergents. Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen. The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

Regulatory properties of potato-Arabidopsis hybrid ADP-glucose pyrophosphorylase

In higher plants, ADP-glucose pyrophosphorylase (ADPGlc-PPase) is a heterotetrameric enzyme comprised of two small and two large subunits. Potato-Arabidopsis hybrid ADPGlc-PPases were generated and their regulatory properties analyzed. We show that ADPGlc-PPase subunits from two different species can interact, producing active enzymes with new regulatory properties. Depending on the subunit combinations, hybrid heterotetramers showed responses to allosteric effectors [3-phosphoglycerate (3-PGA) and Pi] in the micromolar or millimolar range. While hybrid potato small subunit (PSS) and the Arabidopsis large subunit APL1 showed an extremely sensitive response to 3-PGA and Pi, hybrid PSS/Arabidopsis APL2 was very insensitive to them. Intermediate responses were determined for other subunit combinations.