Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

Interaction of cytochrome b5 with surfactant vesicles.

Lysates of protoplasts from the endosperm of developing grains of wheat (Triticum aestivum) were fractionated on density gradients of Nycodenz to give amyloplasts. Enzyme distribution on the gradients suggested that: (i) starch synthase and ADP-glucose pyrophosphorylase are confined to the amyloplasts; (ii) pyrophosphate: fructose-6-phosphate 1-phosphotransferase and UDP-glucose pyrophosphorylase are confined to the cytosol; (iii) a significant proportion (23-45%) of each glycolytic enzyme, from phosphoglucomutase to pyruvate kinase inclusive, is in the amyloplast. Starch synthase, ADP-glucose pyrophosphorylase and each of the glycolytic enzymes showed appreciable latency when assayed in unfractionated lysates of protoplasts. No activity of fructose-1,6-bisphosphatase was found in amyloplasts or in homogenates of endosperm. Antibody to plastidic fructose-1,6-bisphosphatase did not react positively, in an immunoblot analysis, with any protein in extracts of wheat endosperm. It is argued that wheat endosperm lacks significant plastidic fructose-1,6-bisphosphatase and that carbon for starch synthesis does not enter the amyloplast as a C-3 compound but probably as hexose phosphate.     

Carbohydrate metabolism in Musa paradisiaca

Considerable variations exist in the content of glucose, fructose, sucrose, starch and protein and in the activities of enzymes involved in carbohydrate metabolism between different parts of the banana plant (Musa paradisiaca). Sucrose synthetase is present in the highest concentration in rootstock and fruit pulp, and sucrose phosphate synthetase in the pseudostem. The highest ratio of the activity of sucrose phosphate synthetase to sucrose synthetase is found in leaves. Acid invertase is present in leaves, leaf-sheath and fruit pulp and is not demonstrable in rootstock and pseudostem. Neutral invertase activity is high in pseudostem and leaf-sheath. Starch phosphorylase is largely concentrated in fruit pulp and rootstock. The maximum activity of ATP:d-phosphoglucose (ADPG) pyrophosphorylase is found in rootstock. β-Amylase is not demonstrable in rootstock and is largely concentrated in leaf-sheath. Hexokinase is most active in rootstock and the lowest in leaves. Acid phosphatase and alkaline phosphatase activity is highest in fruit pulp and pseudostem. Glucosephosphate isomerase is most active in the rootstock and lowest in the leaves.     

Enzymic properties of amyloplasts form suspension cultures of soybean

The aim of this work was to determine which enzymes of carbohydrate metabolism are present in amyloplasts. Protoplasts from 4- to 5-day-old suspension cultures of soybean, Glycine max, were lysed and fractionated on a sucrose gradient. This gave an amyloplast fraction that contained stromal enzymes and was not seriously contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. Studies of this fraction provide evidence that, in soybean cells, starch synthase and ADPglucose pyrophosphorylase are confined to amyloplasts; invertase, sucrose synthetase and UDPglucose pyrophosphorylase are absent from the amyloplast and probably confined to the cytosol; the following enzymes, though predominantly cytosolic, are present in the amyloplasts in activities high enough to mediate the rate of starch synthesis observed in vivo: glyceraldehyde-phosphate dehydrogenase (NAD), triosephosphate isomerase, fructose-1, 6-bisphosphate aldolase, fructose-bisphosphatase, glucosephosphate isomerase and phosphoglucomutase. The pathway from sucrose to starch in non-photosynthetic cells is discussed; particularly the possibility that sucrose is converted to triose phosphate for entry into the amyloplast.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q_A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O₂ evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O₂ evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl chlorophyll - Fru6P fructose-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - Fru-1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - Fru2,6Pase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - PGI phosphoglucose isomerase - Pi inorganic phosphate - Q_A acceptor for photosystem II - Ru1,5bisP ributose-1,5-bisphosphate - SPS sucrose-phosphate synthase     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme

Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.     

Nonstructural carbohydrate metabolism during leaf ageing in tobacco (Nicotiana tabacum)

Nonstructural carbohydrate status and activities of ADP-glucose pyrophosphorylase (EC 2.7.7.27, ADPG pyrophosphorylase) and sucrose phosphate synthase (EC 2.4.1.14, SPS) were determined during ageing of tobacco ( Nicotiana tabacum L., cvs KY 14 and Speight G28) leaves sampled from control plants and from plants that had the apical meristem and subsequent axillary growth removed (detopped plants). Over the 30-day period shoot growth increased much more for control compared to detopped plants, but the increase in root growth was similar for both treatments. Dry matter and leaf area of the individual leaf used for enzyme and metabolite analysis were constant over time for controls but increased 5-fold for detopped plants. Ageing of control leaves was indicated by a progressive loss of chlorophyll and ribulose 1, 5-bisphosphate carboxylase (EC 4.1.1.39, Rubisco) activity; loss of these components was diminished for detopped plants. In contrast to chlorophyll and Rubisco activity, activities of ADPG pyrophosphorylase and SPS remained relatively constant over time for controls. Thus, under normal ageing conditions, changes in activities of ADPG pyrophosphorylase and SPS were not closely associated with changes in the standard senescence indicators chlorophyll and Rubisco activity. The activities of ADPG pyrophosphorylase and SPS were enhanced, relative to controls, within 6 days after applying the detopping treatment and activities remained high for the duration of the 30-day period. Detopping also led to increased concentrations of starch and sucrose, but the increases were not well correlated with changes in enzyme activities. The data indicated that the leaves of detopped plants functioned as both source leaves, with enhanced ability to synthesize carbohydrate, and sink leaves, with enhanced growth. Therefore, activities of ADPG pyrophosphorylase and SPS were more responsive to changes within an individual leaf than to changes in whole plant growth.     

Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize

Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP-glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts.     

Enzymes of carbohydrate metabolism in the developing endosperm of maize

A number of enzymes presumably implicated in starch synthesis were assayed at various stages of endosperm development ranging from 8 days to 28 days after pollination. Activity for invertase, hexokinase, the glucose phosphate isomerases, the phosphoglucomutases, phosphorylase I, uridine diphosphate glucose pyrophosphorylase, and the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase was present at the earliest stage of development (8 days) studied. Activity was detectable for phosphorylase III, the soluble adenosine diphosphate glucose-starch glucosyltransferase, adenosine diphosphate glucose pyrophosphorylase, and sucrose-uridine diphosphate glucosyltransferase at 12 days. For phosphorylase II and cytidine diphosphate glucose pyrophosphorylase, activity was first detectable at the 14- and 16-day stages, respectively. Rapid increases in starch content are observed prior to detectable activity for adenosine diphosphate glucose pyrophosphorylase, the soluble adenosine diphosphate glucose-starch glucosyltransferase and phosphorylases II and III. For all enzymes, except invertase, activity per endosperm rises to a peak at 22 or 28 days. Greatest activity for invertase is found at 12 days with a steady decline thereafter. The pattern of invertase activity in comparison with that of sucrose-uridine diphosphate glucosyltransferase supports previous suggestions, that the latter plays a key role in the conversion of sucrose to starch. In addition to phosphorylases I, II, and III, multiple forms of glucosephosphate isomerase and phosphoglucomutase were detected.     

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the sink regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO₂. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.Abbreviations Fru 1,6bisP fructose-1,6-bisphosphate - Fru 1,6bisPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc 1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFK phosphofructokinase - PEP phosphoenolpyruvate - PFP pyrophosphate:fructose-6-phosphate phosphotransferase - PGA glycerate-3-phosphate - PK pyruvate kinase - Pi inorganic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SPS sucrose-phosphate synthase - triose-P triose-phosphates     

Limitation of CO(2) Assimilation and Regulation of Benson-Calvin Cycle Activity in Barley Leaves in Response to Changes in Irradiance, Photoinhibition, and Recovery

The response of the Benson-Calvin cycle to changes in irradiance and photoinhibition was measured in low-light grown barley (Hordeum vulgare) leaves. Upon the transition from the growth irradiance (280 micromoles per square meter per second) to a high photoinhibitory irradiance (1400 micromoles per square meter per second), the CO₂ assimilation rate of the leaves doubled within minutes but high irradiance rapidly caused a reduction in quantum efficiency. Following exposure to high light the activities of NADP-malate dehydrogenase and fructose-1,6-bisphosphatase obtained near maximum values and the activation state of ribulose-1,5-bisphosphate carboxylase increased. The activity of the latter remained constant throughout the period of photoinhibitory irradiance, but the increase in the activities of fructose-1,6-bisphosphatase and NADP-malate dehydrogenase was transient decreasing once more to much lower values. This suggests that immediately following the transition to high light reduction and activation of redox-modulated enzymes occurred, but then the stroma became relatively oxidized as a result of photoinhibition. The leaf contents of glucose 6-phosphate and fructose 6-phosphate increased following exposure to high light but subsequently decreased, suggesting that following photoinhibition sucrose synthesis exceeded the rate of carbon assimilation. The ATP content attained a constant value much higher than that in low light. During photoinhibition the glycerate 3-phosphate content greatly increased while ribulose-1,5-bisphosphate decreased. The fructose-1,6-bisphosphate and triose phosphate contents increased initially and then remained constant. During photoinhibition CO₂ assimilation was not limited by ribulose-1,5-bisphosphate carboxylase activity but rather by the regeneration of the substrate, ribulose-1,5-bisphosphate, related to a restriction on the supply of reducing equivalents.     

Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules

Nitrogen is exported in the form of ureides or amides from the nodules in pulse crops. In order to understand the carbon metabolism in ureide and amide exporting nodules, activities of enzymes involved in glucose metabolism were compared in cytosolic and bacteroidal fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules during development. Activities of hexokinase, fructokinase, phosphoglucomutase, fructose-1,6-bisphosphatase, phosphohexose isomerase and UDP-glucose pyrophosphorylase were detected in cytosolic fraction of nodules of both the crops during development. Out of these enzymes, specific activity of phosphohexose isomerase was the highest in nodules of both the crops, in comparison with other enzymes. In comparison with mungbean, activities of various enzymes were less in cytosolic fraction of lentil. Activities of hexokinase, fructokinase, phosphoglucomutase were present only in cytosolic fraction of mungbean (Vigna radiata L.), however, low activity of these enzymes was also observed in lentil (Lens culinaris L.) bacteroids. Activities of phosphohexose isomerase and fructose-1,6-bisphosphatase were higher in bacteroids of lentil, as compared to mungbean during early nodule development, but this pattern was reversed with progress of crop development. Higher activities of phosphoglucomutase and fructose-1,6-phosphatase in mungbean cytosolic fraction could lead to increased flow of carbon towards pentose phosphate pathway.     

Evidence for control of carbon partitioning by fructose 2,6-bisphosphate in spinach leaves

Excision of spinach (Spinacia oleracea L.) leaves had no effect on photosynthetic rates, but altered normal carbon partitioning to favor increased formation of starch and decreased formation of sucrose. The changes were evident within 2 hours after excision. Concurrently, leaf fructose-2,6-bisphosphate content increased about 5-fold (from 0.1 to 0.5 nanomoles per gram fresh weight). The activities of sucrose-P synthase and cytoplasmic fructose 1,6-bisphosphatase in leaf extracts remained constant during the time period tested. It is postulated that the rise in fructose 2,6-bisphosphate was responsible for the change in carbon partitioning.     

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO₃⁻ per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O₂ per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes

Sugar beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically for 2 weeks in growth chambers with two levels of orthophosphate (Pi) supplied in half strength Hoagland solution. Low-P plants were supplied with 1/20th of the Pi supplied to control plants. With low-P treatment, the acid soluble leaf phosphate and total leaf P decreased by about 88%. Low-P treatment had a much greater effect on leaf area than on photosynthesis. Low-P decreased total leaf area by 76%, dry weight per plant by 60%, and the rate of photosynthesis per area at light saturation by 35%. Low-P treatment significantly decreased the total extractable activity of phosphoglycerate kinase (by 18%) and NADP-glyceraldehyde-3-phosphate dehydrogenase (by 16%), but did not decrease the total activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) and ribulose-5-phosphate kinase. Low-P treatment decreased the initial activities of three rate-limiting Calvin cycle enzymes, but had no effect on the initial activity of RuBPCase. Furthermore, low-P treatment significantly increased the total extractable activities of fructose-1,6-bisphosphatase (by 61%), fructose-1,6-bisphosphate aldolase (by 53%), and transketolase (by 46%). The results suggest that low-P treatment affected photosynthetic rate through an effect on RuBP regeneration rather than through RuBPCase activity and that the changes in Calvin cycle enzymes with low-P resulted in an increased flow of carbon to starch.     

Enzymes Catalyzing the Reversible Conversion of Fructose-6-Phosphate and Fructose-1,6-Bisphosphate in Maize (Zea mays L.) Kernels

The significance of the glycolytic and gluconeogenic conversion of fructose-6-phosphate and fructose-1,6-bisphosphate on sugar metabolism was investigated in maize (Zea mays L.) kernels. Maximum extractable activities of the pyrophosphate (PPi) dependent phosphofructokinase, fructose-1,6-bisphosphatase, and the ATP-dependent phosphofructokinase were measured in normal and four maize genotypes, which accumulate relatively more sugars and less starch, to determine how these enzymes are affected by the genetic lesions. Normal endosperm accumulated more dry matter than the high sugar/low starch genotypes, but protein contents did not differ greatly among the genotypes. Mutation of several starch biosynthetic enzymes had little impact on the activities of PPi-dependent phosphofructokinase, fructose-1,6-bisphosphatase, and ATP-dependent phosphofructokinase, despite the altered capacity of the cell to synthesize starch. The PPi-dependent phosphofructokinase appeared to be more active toward glycolysis in all genotypes studied. Activity of the PPi-dependent phosphofructokinase in shrunken (low sucrose synthase genotype) did not differ from the activity in other genotypes, suggesting that the gluconeogenic production of PPi may not be the primary role of the enzyme. As expected, shrunken kernels contained more sugars and less starch than normal kernels throughout kernel development except at the very early stages. Developmental profiles of normal kernels also showed marked changes in the PPi-dependent phosphofructokinase activity, whereas the level of ATP-dependent phosphofructokinase activity remained relatively steady during kernel development. In addition, the ATP-dependent phosphofructokinase, and not the PPi-dependent phosphofructokinase, appeared to correlate more closely with respiration rate. These findings suggest that glycolysis catalyzed by the ATP-dependent phosphofructokinase may serve primarily to support energy production, and glycolysis catalyzed by the PPi-dependent phosphofructokinase may contribute mainly to generation of biosynthetic intermediates.     

Enzyme activities associated with developing wheat(Triticum aestivum L.) grain amyloplasts

Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P₂ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P₂ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.