Activities of enzymes of sugar metabolism in cold-stored tubers of Solanum tuberosum

Storage of tubers of Solanum tuberosum at 10° or 2° for 15 days did not alter significantly the maximum catalytic activities of sucrose phosphate synthetase, sucrose synthetase, glucose-6-phosphate dehydrogenase, aldolase, and glyceraldehydephosphate dehydrogenase. The temperature coefficients of phosphofructokinase, glyceraldehydephosphate dehydrogenase, and pyruvate kinase from the tubers were shown to be higher between 2° and 10° than between 10° and 25°. The rate of sugar accumulation at 2° exceeded the activity of sucrose synthetase but was less than that of sucrose phosphate synthetase. It is suggested that sucrose accumulation at 2° is catalysed by sucrose phosphate synthetase, is not due to changes in the maximum catalytic activities of any of the above enzymes, but may be due, in part, to the susceptibility of key glycolytic enzymes to cold.     

The purification and properties of sucrose-phosphate synthetase from spinach leaves: the involvement of this enzyme and fructose bisphosphatase in the regulation of sucrose biosynthesis

The properties of spinach leaf sucrose-phosphate synthetase (EC 2.4.1.14) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) have been studied. These two enzymes have been considered to be important in the control of sucrose synthesis. Sucrose-phosphate synthetase from leaf tissue has not been studied in detail previously and we report a technique for purifying this enzyme 50-fold by chromatography on AH-Sepharose 4B. This method frees the enzyme from contaminants which interfere with assay procedures with little or no loss of activity. The partially purified enzyme has a K_m for UDP-glucose of 7.1 mm and for fructose 6-phosphate of 0.8 mm. Fructose 1,6-bisphosphate, inorganic phosphate and UDP are strong inhibitors. The inhibition patterns of these suggest that the enzyme operates either by an ordered bi-bi or a Theorell-Chance mechanism. Partially purified cytosolic fructose-1,6-bisphosphatase is not only inhibited by AMP as previously reported, but is also inhibited by fructose 6-phosphate and UDP. From our observations, we conclude that sucrose biosynthesis is indeed controlled through these two enzymes and it appears that the rate of sucrose synthesis is largely dependent upon the supply of triose phosphate and ATP from the chloroplast.     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.     

Sorbitol metabolism by apple seedlings

The aims of this work were to compare the roles of sorbitol and sucrose in seedlings of Malus domestica, to discover which tissues synthesize sorbitol and which break it down, and to examine these tissues for enzymes of sorbitol metabolism. The detailed distribution of label was determined after supplying intact seedlings with ¹⁴CO₂, and excised parts of seedlings with [U-¹⁴C]fructose and [U-¹⁴C]sorbitol. The results showed that appreciable synthesis of sorbitol occurred only in the leaves but did not depend directly on photosynthesis. All tissues examined metabolized sorbitol but metabolism was extensive only in root apices, and in leaves which had been kept in the dark. The above experiments suggest that sorbitol supplements but does not replace sucrose. Extracts of apple leaves showed no trace of either a polyol or a polyol phosphate dehydrogenase but did exhibit sorbitol-6-phosphate phosphatase activity. A limited number of experiments with extracts of the blades of Laminaria digitata indicated that they contained mannitol-1-phosphate phosphatase and mannitol dehydrogenase.     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

Presence of Nonoxidative Enzymes of the Pentose Phosphate Shunt in Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-¹⁴C]ribose into CO₂ and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.     

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 metabolism of glucose by heterocysts and vegetative cells of Anabaena cylindrica

Whole filaments of autotrophically grown Anabaena cylindrica and heterocysts isolated from them will assimilate and metabolise exogenous glucose. Radiorespirometric experiments suggest the operation of the pentose phosphate pathway. Glucose-6-phosphate and 6-phosphogluconate dehydrogenase are present in heterocysts at 6–8 times the levels found in vegetative cells whereas enzymes of the reductive pentose phosphate and glycolytic pathways are barely or not detectable. Glucose-6-phosphate dehydrogenase in vegetative cells, but not in heterocysts is subject to inhibition by ribulose diphosphate.     

Selective Inhibition by Actinomycin D of the Synthesis in Photosynthetic and Non-photosynthetic Enzymes During the Greening of Etiolated Bean Leaves

The effect of actinomycin D on the synthesis of the photosynthetic apparatus during illumination of etiolated leaves of Phaseolus vulgaris was studied. The increase of chlorophyll content and of the activities of some photosynthetic enzymes (NADPH diaphorase, ferredoxin, NADP⁺ glyceraldehyde-3-phosphate dehydrogenase) was compared with simultaneous measurements of the level of other enzymes not considered associated with photosynthesis (ornithine transcarbamylase, glucose-6-phosphate dehydrogenase, NAD⁺ glyceraldehyde-3-phosphate dehydrogenase).     

Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

Mannitol Metabolism in Lentinus edodes, the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.     

Isozymes of the glycolytic enzymes in endosperm from developing castor oil seeds

Ion filtration chromatography on diethylaminoethyl-Sephadex A-25 has been used to separate two isozymes each of triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, enolase, and phosphoglycerate mutase from homogenates of developing castor oil (Ricinus communis L. cv. Baker 296) seeds. Crude plastid fractions, prepared by differential centrifugation, were enriched in one of the isozymes, whereas the cytosolic fractions were enriched in the other. These data (and data published previously) indicate that plastids from developing castor oil seeds have a complete glycolytic pathway and are capable of conversion of hexose phosphate to pyruvate for fatty acid synthesis. The enzymes of this pathway in the plastids are isozymes of the corresponding enzymes located in the cytosol.     

Presence of nonoxidative enzymes of the pentose phosphate shunt in Tetrahymena.

Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-14C]ribose into CO2 and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.     

Characterization of the fatty acid-sensitive glucose 6-phosphate dehydrogenase from Pseudomonas cepacia.

The adenosone 5'-triphosphate-insensitive glucose 6-phosphate dehydrogenase from Pseudomonas cepacia has been found to be strongly inhibited by long-chain fatty acids and their acyl coenzyme A esters, suggesting that an important role of this isoenzyme might be to provide reduced nicotinamide adenine dinucleotide phosphate for reductive steps in fatty acid synthesis. The enzyme, which has been redesignated the fatty acid-sensitive glucose 6-phosphate dehydrogenase, has been purified to homogeneity using affinity chromatography with nicotinamide adenine dinulceotide phosphate-substituted Sepharose as a key step in the purification. The purified preparations were used to study the immunological properties and subunit composition of the enzyme and its relationship to the adenosine 5'-triphosphate-sensitive glucose 6-phosphate dehydrogenase present in extracts of P. cepacia. Although both enzymes were found to be composed of similar size subunits of about 60,000 daltons, immunological studies failed to demonstrate any antigenic similarity between them. Studies of the sedimentation behavior of the fatty acid-sensitive enzyme in sucrose gradients indicated that its apparent molecular weight is increased in the presence of glucose 6-phosphate and suggest that it may exist in an aggregated state in vivo. Palmitoyl coenzyme A, which strongly inhibited the enzyme, failed to influence its sedimentation behavior.     

Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves

The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.     

Leaf Carbon Metabolism and Metabolite Levels during a Period of Sinusoidal Light

Photosynthesis rate, internal CO₂ concentration, starch, sucrose, and metabolite levels were measured in leaves of sugar beet (Beta vulgaris L.) during a 14-h period of sinusoidal light, which simulated a natural light period. Photosynthesis rate closely followed increasing and decreasing light level. Chloroplast metabolite levels changed in a manner indicating differential activation of enzymes at different light levels. Starch levels declined during the first and last 2 hours of the photoperiod, but increased when photosynthesis rate was greater than 50% of maximal. Sucrose and sucrose phosphate synthase levels were constant during the photoperiod, which is consistent with a relatively steady rate of sucrose synthesis during the day as observed previously (BR Fondy et al. [1989] Plant Physiol 89: 396-402). When starch was being degraded, glucose 1-phosphate level was high and there was a large amount of glucose 6-phosphate above that in equilibrium with fructose 6-phosphate, while fructose 6-phosphate and triose-phosphate levels were very low. Likewise, the regulatory metabolite, fructose, 2,6-bisphosphate was high, indicating that little carbon could move to sucrose from starch by the triose-phosphate pathway. These data cast doubt upon the feasibility of significant carbon flow through the triose-phosphate pathway during starch degradation and support the need for an additional pathway for mobilizing starch carbon to sucrose.     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.