Sucrose synthase of soybean nodules

Sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyl transferase, EC 2.4.1.13) has been purified from the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules. The native enzyme had a molecular weight of 400,000. The subunit molecular weight was 90,000 and a tetrameric structure is proposed for soybean nodule sucrose synthase. Optimum activity in the sucrose cleavage and synthesis directions was at pH 6 and pH 9.5 respectively, and the enzyme displayed typical Michaelis-Menten kinetics. Soybean nodule sucrose synthase had a high affinity for UDP (K_m, 5 micromolar) and a relatively low affinity for ADP (apparent K_m, 0.13 millimolar) and CDP (apparent K_m, 1.1 millimolar). The K_m for sucrose was 31 millimolar. In the synthesis direction, UDPglucose (K_m, 0.012 millimolar) was a more effective glucosyl donor than ADPglucose (K_m, 1.6 millimolar) and the K_m for fructose was 3.7 millimolar. Divalent cations stimulated activity in both the cleavage and synthesis directions and the enzyme was very sensitive to inhibition by heavy metals.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.     

Sucrose Metabolism at Three Leaf Development Stages in Bean Plants

Sucrose metabolism was studied at three leaf development stages in two Phaseolus vulgaris L. cultivars, Tacarigua and Montalban. The changes of enzyme activities involved in sucrose metabolism at the leaf development stages were: (1) Sink (9-11 % full leaf expansion, FLE): low total sucrose phosphate synthase (SPS) activity, and higher acid invertase (AI) activity accompanied by low sucrose synthase (SuSy) synthetic and sucrolytic activities. (2) Sink to source transition (40-47 % FLE): increase in total SPS and SuSy activities, decrease in AI activity. (3) Source (96-97 % FLE): high total SPS activity, increased SuSy activities, decreased AI activity. The hexose/sucrose ratio decreased from sink to source leaves in both bean cultivars. The neutral invertase activity was lower than that of AI; it showed an insignificant decrease during the sink-source transition.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

Evidence for the uptake of sucrose intact into sugarcane internodes

Application of [¹⁴C]fructosyl sucrose was used to determine whether sucrose cleavage was necessary for sucrose uptake by sugarcane (Saccharum spp.) internode tissue. Although approximately 25% of ¹⁴C in the apoplast was present as fructose, indicating some sucrose cleavage, less than 15% of the label was randomized in the sucrose that remained in the tissue after a 30 minute osmoticum rinse. This is insufficient to support cleavage and resynthesis as the sole sucrose transport scheme. The lack of randomization of label between the glucose and fructose moieties of the sucrose molecule was taken as presumptive evidence that sucrose does not have to be cleaved prior to uptake by parenchyma cells in sugarcane internode tissue.     

Properties of a Partially Purified Nucleoside Triphosphatase (NTPase) from the Chloroplast Envelope of Pea

The Mg-nucleoside triphosphatase activity associated with the inner envelope membrane of the pea chloroplast is comprised of at least two components, a major activity that is sensitive to vanadate and sodium fluoride and a minor insensitive activity. The vanadate/fluoride sensitive activity has been partially purified (about 35-fold) from Triton X-100 solubilized membranes by DEAE-Sephadex chromatography and sucrose density gradient centrifugation. The partially purified enzyme resembles the membrane-bound activity in requiring either Mg²⁺ or Mn²⁺, having a broad specificity for nucleoside triphosphates, having a K_m for ATP of 0.18 millimolar, and being inhibited by N-ethylmaleimide, but insensitive to sodium azide and dicyclohexylcarbodiimide. The partially purified enzyme obtained after sucrose gradient centrifugation has a markedly increased sensitivity to inhibition by inorganic pyrophosphate compared with the less pure enzyme. Pyrophosphate is not a substrate of either the membrane-bound or partially purified enzyme.     

Effect of deglycosylation on the properties of thermophilic invertase purified from the yeast Candida guilliermondii MpIIIa

Invertase from Candida guilliermondii MpIIIa was purified and biochemically characterized. The purified enzyme (INV3a-N) is a glycoprotein with a carbohydrate composition comprising nearly 74% of its total molecular weight (MW) and specific activity of 82,027 U/mg of protein. The enzyme displayed optimal activity at pH 5.0 and 65 ˚C. The Km and V_max values for INV3a-N were 0.104 mM and 10.9 μmol/min/mg of protein, respectively, using sucrose as the substrate. The enzyme retained 50% and 20% of its maximal activity after 168 h and 30 days, respectively, at 50 ˚C. INV3a-N was fully active at sucrose concentrations of 400 mM and the activity of the enzyme dropped slowly at higher substrate concentration. Interestingly, the deglycosylated form of INV3a-N (INV3a-D) displayed 76–92% lower thermostability than that of INV3a-N at all temperatures assayed (50–70 ˚C), and was inhibited at sucrose concentrations of 200 mM. Findings here indicate glycosylation plays an important role, not only in the thermostability of INV3a-N, but also in the inhibition of the enzyme by sucrose. Since the enzyme is active at high sucrose concentrations, INV3a-N may be considered a suitable candidate for numerous industrial applications involving substrates with high sugar content or for improvement of ethanol production from cane molasses.     

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K_m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K_m for UDP-glucose = 1·9 mM). Values for V_max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro. The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.     

Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves : estimation by alternate substrate utilization

The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1′-deoxy-1′-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS. [¹⁴C]Sucrose and [¹⁴C]FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when [¹⁴C]sucrose was supplied than when [¹⁴C]FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.     

Constitutive mutants for dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Constitutive mutants for dextransucrase were isolated from cells of Leuconostoc mesenteroides NRRL B-512F by treatment with N-methyl-N′-nitro-N-nitrosoguanidine, growing on an agar plate containing glucose as a carbon source and overlaying a soft agar with sucrose and tetracycline. These mutants were able to produce the enzyme in a liquid media containing sugars other than sucrose, such as glucose, fructose and maltose, without simultaneous synthesis of dextran. The enzyme activity of one mutant strain, SH 3002, was 2- to 3-fold higher than that of the wild strain grown on sucrose. When the concentration of glucose in the medium was increased from 2 to 4%, a 1.7-fold increase of enzyme activity was obtained for the mutant, whereas only a slight increase of the activity was observed on sucrose for both the wild strain and the mutant.     

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH₄)₂SO₄ fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K_m values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50°C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.     

Production of 1-Kestose in Transgenic Yeast Expressing a Fructosyltransferase from Aspergillus foetidus

Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase (1-SST) from Aspergillus foetidus has been purified and subjected to N-terminal amino acid sequence determination. The enzyme is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular mass of 180 kDa as judged from mobility in seminative acrylamide gels. The enzyme catalyzes fructosyl transfer from sucrose to sucrose producing glucose and 1-kestose. Oligosaccharides with a higher degree of polymerization are not obtained with sucrose as the substrate. The cDNA encoding the A. foetidus 1-SST has been cloned and sequenced. Sequence homology was found to be highest to levanases, but no hydrolytic activity was observed when levan was incubated with the enzyme. Expression of the cloned gene in an invertase-deficient mutant of Saccharomyces cerevisiae resulted in 1-kestose production, with 6-kestose and neokestose being side products of the reaction. Products were well distinguishable from those formed by yeast transformants expressing a cytosolic invertase.     

Sucrose metabolism and accumulation in developing fruit of Cucumis

Sucrose, reducing sugars and starch content were measured in developing cucumber (C. sativus cv Delilah), sweet melon (C. melon cv Galia and cv Noy Yizre'el) and non-sweet melon (C. melo cv Bird's Nest) fruit. Sweet melon were characterized by accumulation of sucrose in the maturing fruit, while cucumber and non-sweet melon had a low sucrose content at all stages studied. Soluble acid invertase activity (EC 3.2.1.26) dramatically decreased in sweet melon, concomitant with the onset of sucrose accumulation. Significant activity of soluble acid invertase was retained in mature cucumber and non-sweet melon. Insoluble acid invertase, determined not to be an artifact of extraction, constituted a significant portion of total invertase activity (ca 25% in young sweet melon and ca 50% in young cucumber). In sweet melon sucrose synthase activity (EC 2.4.1.13), measured in both the cleavage and synthesis direction, increased during the sucrose accumulation period. The results are discussed in terms of the roles of invertase and sucrose synthase in sucrose accumulation in Cucumis.     

Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division.     

Enzymic Components of Sucrose Accumulation in the Wild Tomato Species Lycopersicon peruvianum

Sugar and soluble solids content and invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13), and sucrose phosphate synthase (EC 2.4.1.14) enzyme activities were measured throughout fruit development in tomato (Lycopersicon esculentum Mill.) and the green fruited species Lycopersicon peruvianum. Fruit of L. peruvianum accumulated predominantly sucrose, in contrast with hexose accumulation, which is characteristic of L. esculentum. The percentage of soluble solids in ripe L. peruvianum fruit was more than twice that present in L. esculentum and attributed primarily to the high level of sucrose accumulated in L. peruvianum. Low levels of invertase and sucrose synthase activity were associated with the period of significant sucrose accumulation and storage in L. peruvianum. Increased sucrose phosphate synthase activity was observed during the latter stages of fruit development in sucrose-accumulating fruit but was not coincident with maximum rates of sucrose accumulation.     

Regulation of invertase levels in Avena stem segments by gibberellic Acid, sucrose, glucose, and fructose

Gibberellic acid and sucrose play significant roles in the increases in invertase and growth in Avena stem segments. About 80% of invertase is readily solubilized, whereas the rest is in the cell wall fraction. The levels of both types of invertase change in a similar manner in the response to gibberellic acid and sucrose treatment. The work described here was carried out with only the soluble enzyme. In response to a treatment, the level of invertase activity typically follows a pattern of increase followed by decrease; the increase in activity is approximately correlated with the active growth phase, whereas the decrease in activity is initiated when growth of the segments slows. A continuous supply of gibberellic acid retards the decline of enzyme activity. When gibberellic acid was pulsed to the segments treated with or without sucrose, the level of invertase activity increased at least twice as high in the presence of sucrose as in its absence, but the lag period is longer with sucrose present. Cycloheximide treatments effectively abolish the gibberellic acid-promoted growth, and the level of enzyme activity drops rapidly. Decay of invertase activity in response to cycloheximide treatment occurs regardless of gibberellic acid or sucrose treatment or both, and it is generally faster when the inhibitor is administered at the peak of enzyme induction than when given at its rising phase. Pulses with sucrose, glucose, fructose, or glucose + fructose elevate the level of invertase significantly with a lag of about 5 to 10 hours. The increase in invertase activity elicited by a sucrose pulse is about one-third that caused by a gibberellic acid pulse given at a comparable time during mid-phase of enzyme induction, and the lag before the enzyme activity increases is nearly twice as long for sucrose as for gibberellic acid. Moreover, the gibberellic acid pulse results in about three times more growth than the sucrose pulse. Our studies support the view that gibberellic acid, as well as substrate (sucrose) and end products (glucose and fructose), play a significant role in regulating invertase levels in Avena stem tissue, and that such regulation provides a mechanism for increasing the level of soluble saccharides needed for gibberellic acid-promoted growth.     

Purification and properties of invertase from a glutamate-producing bacterium

Invertase was purified from the cell extracts of the glutamic acid bacterium (Brevibacterium divaricatum) by ammonium sulfate fractionation, batch theatment with DEAE-cellulose, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was proved to be almost homogeneous by polyacrylamide gel electrophoresis.The molecular weight of the enzyme was estimated to be 92,000 by both gel filtration and SDS-polyacrylamide gel electrophoresis methods. The optimum pH and temperature for the activity were 6.8 and 40°C. The enzyme was highly specific to sucrose as substrate, having only 10% as much activity toward raffinose as that toward sucrose, and being inert toward other disaccharides: maltose, trehalose, lactose, melibiose and cellobiose. The K_m value for sucrose was 0.19 M. The enzyme required phosphate or arsenate ions for activity. Monovalent or divalent Cu ions and sulfhydryl reagents inhibited the enzyme.     

Evidence for the involvement of sucrose phosphate synthase in the pathway of sugar accumulation in sucrose-accumulating tomato fruits

To better understand the mechanism of sugar unloading and sugar concentration in hexose- and sucrose-accumulating tomato fruits (Lycopersicon chmielewskii and L. esculentum, respectively) and to determine the causes of the late accumulation of sucrose present in sucrose-accumulating tomato fruits, the assimilation of [³H](fructosyl)-sucrose was studied. Key enzymes involved in carbohydrate metabolism were also assayed. The results demonstrated that the low level of sucrose present in young fruits accumulates directly without undergoing hydrolysis, suggesting a symplastic pathway for sucrose unloading. By contrast, the large quantity of the sucrose present in ripe sucrose-accumulating fruits originates from hydrolysis and resynthesis, suggesting an apoplastic pathway for sucrose unloading. The increase in sucrose level observed in sucrose-accumulating fruits is associated with a gradual decline in invertase activity and an increase in sucrose phosphate synthase activity. This latter enzyme seems to play a key biochemical role in the accumulation of sucrose and the establishment of a high sugar content in tomato fruits.