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Foi isolada de fruta deteriorada uma linhagem de klebsiella sp que transforma sacarose em palatinose. Foi verificado que a linhagem produz uma glicosiltransferase intracelular que transforma sacarose para palatinose. A enzima de Klebsiella sp foi purificada por fracionamento com sulfato de amônio e cromatografia em colunas de DEAE-Sephadex A-50 e CM-celulose. A enzima purificada apresentou ótima atividade a 35ºC e na faixa de pH 6,0 a 6,5. A enzima foi inibida por Hg2+ e Ag+, mas näo foi inibida por p-cloromercuribenzoato. Os valores de km e Vmax da enzima purificada foram respectivamente 120mM e 110µg palatinose formada por minuto por ml de enzima para o substrato sacarose. O peso molecular da enzima foi estimado em 74.000 Da. A enzima converteu 4 por cento (p/w) de sacarose para isomaltulose com uma eficiência de 86 por cento a 25ºC, pH 6,5

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Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants. The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis. A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6-biphosphate in source leaves. This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO2. Measuring photosynthetic carbon fluxes by labelling leaf discs with 14CO2 revealed a 53-65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18-24%. The flux into the anionic and cationic fraction was not altered. Despite these changes steady-state sucrose concentrations were not effected in source leaves from transgenic plants. Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night. This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark. Accordingly, plant growth and potato tuber yield remained unaltered. From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy. Whether the same holds true for field conditions remains to be elucidated.

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A produçäo de micélio seco de Metarhizium anisopliae (CG 46, isolado da cigarrinha das pastagens Deois flavopicta) foi investigada. Várias fontes de carbono (dextrose e sacarose) e nitrogênio (extrato e água de levedura) foram testadas em cultivos em incubador rotativo (150 rpm). As melhores fontes de carbono e nitrogênio foram sacarose (4 por cento) e extrato de levedura (1 por cento), respectivamente. A concentraçäo celular (massa seca) e pH variaram de acordo com a concentraçäo inicial de conídios. A maior massa seca foi obtida com concentraçöes altas de conídios (> 10x7 conídios por ml). No fermentador, com meio de cultura à base de sacarose (4 por cento) e extrato de levedura (1 por cento), às 72 h de cultivo, a massa final foi 9.05 g/l com uma produtividade de 0,13 g.l(-1).h(-1). O consumo de açucar a 72 h de cultivo foi cerca de 69 por cento. Após rehidrataçäo (72 h, 25oC), o fungo produziu 1,63 x 10(10) conídios/g de micélio seco. A viabilidade dos conídios produzidos foi superior a 90 por cento. A utilizaçäo de água de levedura pode ser facilmente preparada a partir de sub-produtos de destilarias de álcool

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Rate of net CO2 exchange and activities of the key enzymes of fru-2,6-P2, sucrose and starch synthesis and levels of certain intermediates of Calvin cycle were determined in Brassica pods at different stages of their development. The rate of net CO2 exchange, activities of FBPase, UDPG-pyrophosphorylase and SPS, and the contents of 3-PGA, DHAP, RuBP and UDPG increased up to day 21 after anthesis followed by a continuous decrease thereafter. However the content of fru-6-P started decreasing only after 28 days of anthesis. Changes in the levels of fru-2,6-P2 were closely associated with the changes in F6P 2-kinase activity rather than with F2,6-P2ase activity. Similarly, activities of ADPG-pyrophosphorylase and ADPG-starch synthetase closely followed the pattern of starch accumulation in pod tissues. These observations suggest that during the early phase of pod development (up to 21 days after anthesis), which is also the active phase for pod photosynthesis, carbon is mainly utilised for sucrose synthesis and that during the later phase of pod development (from day 21 to 42 after anthesis), there is shift in metabolic path of carbon from sucrose to starch.

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We extend the analysis of unbranched chains (preceding paper) to large parameter changes in branched systems using linear kinetic assumptions. More complex relationships between flux control coefficients and deviation indices are established. In particular, the deviation index in such systems depends on more than one control coefficient as well as on the magnitude of the enzyme change. Non-additivity of the indices is the general rule. Combined changes of groups of enzymes, whether co-ordinate or not, have also been formulated. Control coefficients can be estimated from a small number of independent large-change experiments. Alternatively, the amplification factors can be calculated given the knowledge of the control coefficients. A 'case study' using published data is presented. The movement of intermediate metabolites as a consequence of large parameter changes can be dealt with in a similar manner. Experimental methods for showing the admissibility of assuming the simplifying assumptions used are summarised. Some simulation studies show possible limits of the application of the approach and some aspects of the general, non-linear, case are discussed. It is concluded that, although metabolic systems are in principle non-linear, many behave, in practice, as quasi-linear systems. The relationships established between deviation indices and control coefficients therefore provide a practical way of predicting the effects of large-scale changes in parameters for many metabolic systems.

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Candida stellatoidea is classically distinguished from C. albicans by the ability of the latter species to assimilate sucrose. We show here that sucrose-positive revertants of C. stellatoidea type II are readily isolated and that C. stellatoidea type II strains probably resulted from a mutation in the sucrase gene of C. albicans. The revertants were not laboratory contaminants, as determined by restriction fragment length polymorphism analysis and retention of an auxotrophic marker. The reversion of three tested strains was accompanied by 16 to 110-fold increases in expression of a sucrase/alpha-glucosidase but not an invertase, with a Km for sucrose of about 1 mM. The enzyme activity was assayable in intact cells. The drastically increased expression of such an enzyme would allow extracellular sucrose hydrolysis and assimilation of the monosaccharide products.

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This paper describes cell-free enzymatic syntheses of sucrose and trehalose using partially-purified preparations of sucrose and trehalose synthetase. The coupling of the regeneration of uridine-5'-diphosphoglucose (UDP-Glc) with synthesis of the disaccharide offers a practical route to millimol quantities of these carbohydrates. The syntheses used pyruvate kinase, UDP-Glc pyrophosphorylase, and inorganic pyrophosphatase, and the regenerated UDP-Glc was cycled approximately 10 times.

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Detached glandular trichome head preparations and epidermal strips with and without trichome heads were used to identify glandular trichome heads as the site of sucrose ester biosynthesis in tobacco. Carbon dioxide in solution as well as sucrose, glucose, and acetate were shown to serve as precursors to both sucrose esters and duvatrienediol diterpenes in detached trichome heads or epidermal strips, and gaseous CO2 was also efficiently utilized by epidermal strips. Thus, glandular heads can biosynthesize these principal exudate components from a molecule as simple as CO2. While formation of duvatriendiols from all precursors tested and conversion of sucrose and glucose to sucrose esters was light dependent, utilization of acetate to label the 6-O-acetyl group of the glucose moiety of sucrose esters occurred equally well in light and dark. The data suggest that CO2 and/or monosaccharides produced in trichome head cells and perhaps that supplied by other epidermal cells can act as carbon sources for sucrose ester and duvatrienediol biosynthesis which occurs in the glandular trichome head.

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The effects of lowering the temperature from 25 degrees C to 2-8 degrees C on carbohydrate metabolism by plant cells are considered. Particular emphasis is placed on the mechanism of cold-induced sweetening in tubers of potato (Solanum tuberosum). Temperatures between 0 and 10 degrees C were shown to cause a marked reduction in the rate of respiration of a wide range of plant tissues. At these temperatures the ability of suspension cultures of soybean (Glycine max), and callus cultures and tubers of potato to metabolize [14C]glucose was appreciably diminished. The detailed distribution of 14C showed that lowering the temperature decreased the proportion of the metabolized [14C]glucose that entered the respiratory pathways and increased the proportion converted to sucrose. Pulse and chase experiments, in which [14C]glucose was supplied to potato tubers at 2 and 25 degrees C, showed that lowering the temperature led to accumulation of label in hexose 6-phosphates, which were subsequently converted to sucrose. The patterns of 14CO2 production from specifically labelled [14C]glucose supplied to soybean suspension cultures and disks of potato tuber suggested that lowering the temperature reduced the activity of glycolysis more than that of the oxidative pentose phosphate pathway. It is argued that the above experiments demonstrate that lowering the temperature not only reduces the rate of carbohydrate metabolism but also alters the relative activities of the different pathways involved. A disproportionate reduction in glycolysis at the lower temperatures is suggested. Mature tubers of many varieties of potato accumulate sucrose and hexose when stored between 2 and 10 degrees C. Starch is the source of carbon for this synthesis of sugar. We could not detect cytosolic fructose-1,6-bisphosphatase in potato tubers and suggest that carbon for sugar synthesis in the cold leaves the amyloplast, not as triose phosphate, but probably as a six-carbon compound. Evidence is presented that phosphofructokinase (EC 2.7.1.11) plays a major role in regulating the entry of hexose 6-phosphates into glycolysis in potato tubers. Phosphofructokinase was purified from potato tubers and shown to consist of four forms. Three of these forms were shown to have higher Q10 values over the range 2-6 degrees C than over the range 12-16 degrees C and are regarded as being cold-labile. No such cold-lability was detected for the key enzymes involved in sucrose synthesis and the oxidative pentose phosphate pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

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Protoplasts from the leaves of wheat, spinach, and barley were found to synthesize [14C]sucrose from 14CO2 at rates comparable with those of the parent tissue. CO2 fixation and sucrose biosynthesis ceased virtually immediately when the light was switched off. The effect of sucrose pretreatment on the rate of de novo sucrose biosynthesis was found to vary with leaf age and with plant species. Protoplasts from young wheat and spinach leaves showed an apparent stimulation of the rate of sucrose biosynthesis after sucrose pretreatment. In protoplasts from mature leaves of spinach, sucrose pretreatment produced inhibition. After sucrose pretreatment protoplasts from mature spinach leaves showed low rates of CO2 fixation, and sucrose biosynthesis compared with controls. Conversely, with protoplasts from mature leaves of wheat and barley, the rate of CO2 fixation was unchanged and there was little or no effect on the rate of sucrose biosynthesis after sucrose pretreatment. Preincubation with sucrose had no effect on the activity of sucrose-phosphate synthetase (EC 2.4.1.14), cytoplasmic fructose-1,6-bisphosphatase (EC 3.1.3.11), or UDPglucose pyrophosphorylase (EC 2.7.7.9) from spinach leaves. It was concluded that there is no direct feedback inhibition of sucrose on the sucrose biosynthetic pathway in leaves of spinach, wheat, and barley. The mechanism of inhibition of sucrose biosynthesis by sucrose in spinach remains to be elucidated.

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Extracellular glucosyltransferase (sucrose:1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) was purified about 10 000-fold from the culture supernatant of Streptococcus mutans 6715. The enzyme preparation was homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing and ultracentrifugation analyses. The specific activity of the enzyme was 34.9 I.U. per mg of protein and the carbohydrate content was less than 1% (w/w). The molecular weight was determined to be 149 000 +/- 5000 by sedimentation equilibrium experiment. The acidic and basic amino acids of the enzyme comprised 29 and 8.4% of total amino acid, respectively, and the isoelectric point was pH 4.1. The enzyme had the optimum pH of 5.5 and the Km value of 2.4 mM for sucrose. The water-soluble glucan, which was de novo-synthesized from sucrose by the purified enzyme, was analyzed by a gas-liquid chromatography-mass spectroscopy and was found to be 1,6-alpha-D-glucan with highly (35%) branched structure of 1,3,6-linked glucose residue.

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Five strains of Streptococcus mutans were grown in continuous culture with either a limited supply or an excess of glucose. Proteins secreted into the extracellular fluid by strains C67-1, 3209 and K1 rapidly catalysed the synthesis of insoluble glucan from sucrose (mutansucrase activity). The culture fluid from strains Ingbritt or C67-25 catalysed the synthesis of soluble glucan (dextransucrase activity) and fructan, but little or no mutansucrase activity was detected. The strains which secreted active mutansucrase readily colonized a smooth hard surface during growth in batch culture and were more cariogenic in pathogen-free rats than those which secreted little mutansucrase activity. There was no similar correlation between fructosyltransferase, dextransucrase or total glucosyltransferase activity and either adherence or cariogenicity. We conclude that the ability to catalyse insoluble glucan synthesis is a major determinant of the cariogenicity of S. mutans strains.

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Extracellular fructosyltransferase (levansucrase; EC 2.4.1.10) production in Actinomyces viscosus T14AV was demonstrated to occur concomitantly with cellular growth. The inhibition of both cellular ribonucleic acid and protein synthesis resulted in no further accumulation of enzyme activity. The antibiotic sodium clofibrate differentially inhibited the production of fructosyltransferase by strain T14AV. Furthermore, the antibiotic preferentially inhibited [14C]acetate incorporation into cellular lipid, but did not affect protein synthesis. In addition, no inhibition of fructosyltransferase production was observed upon the addition of the fatty acid acid synthesis inhibitor cerulenin. On the other hand, extracellular fructosyltransferase production was apparently stimulated in the presence of the cell wall synthesis inhibitors penicillin, amphomycin, and tunicamycin. These results are discussed in terms of the mechanism of extracellular protein production in A. viscosus.

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The light activation of photosynthesis has been investigated in spinach palisade cell protoplasts. (1) After a short induction period, maximal rates of photosynthesis are achieved. (2) [14C]Bicarbonate initially labels anionic compounds in the chloroplast and then in the extrachloroplast compartments. These pools saturate within 2-4 min and radioactivity accumulates mainly in sucrose in the extrachloroplast compartment, in starch and in cationic compounds. (3) Enzymic determinations were made of metabolite levels during the induction period in the chloroplast and extrachloroplast compartments. There is no general build-up of intermediates. Perturbations of individual intermediates occurred, consistent with the activation of specific enzymes. (4) It is suggested that fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase may limit flux in the Calvin cycle during induction. (5) The onset of sucrose synthesis is not accompanied by accumulation of intermediates in the cytosol. It is suggested that sucrose phosphate synthase or sucrose phosphate phosphatase is activated. (6) Measurements of metabolites in whole leaves during a 24 h illumination cycle confirmed that substrates are not depleted during the dark period, and that the onset of photosynthesis is not accompanied by a rise in intermediate levels. (7) It is concluded that the causes of the induction lag in protoplasts can differ from those in isolated chloroplasts.

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The effect of the fatty acid synthesis inhibitor cerulenin on growth and dextransucrase (EC 2.4.1.5) production by Streptococcus mutans 6715 was analyzed. Growth was markedly inhibited by less than 1 microgram of the antibiotic per ml. Under conditions where cerulenin did not inhibit amino acid incorporation into protein but did block acetate metabolism into lipid, the production of extracellular dextransucrase was suppressed. Inhibition was not due to a direct effect of the antibiotic on the enzyme or the lack of release of enzyme from the bacterial cell surface. Gel column chromatography demonstrated that enzyme produced in the presence of cerulenin was highly aggregated, similar to the control enzyme. Although the addition of ysophosphatidylcholine to enzyme which had been synthesized in the presence of cerulenin stimulated glucan formation from sucrose, the increase was not greater than that produced with the control enzyme. The differential inhibition of dextransucrase production by cerulenin indicates that enzyme secretion requires the production of lipid and may reflect the mechanism by whch this enzyme is transported from the bacterial cell.

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Four related hypotheses were tested: 1) substitution of sucrose for starch at moderate levels will significantly elevate blood pressure; 2) most urinary sucrose is endogenous; 3) a change in endogenous sucrose production will alter sodium excretion and blood pressure, and 4) dietary sucrose inhibits endogenous sucrose production. The systolic blood pressures of 25 male rats, 100 days of age, and 25 female rats, 1 year of age, were measured weekly for 8 months. In four experiments, they consumed diets in which 38% of energy came from fat, 15% from protein, 7% from lactose and the remaining 40% from five different sucrose/starch ratios. In experiment 4, a 10% maltose/30% starch diet was fed to one-half the rats fed sucrose in experiment 3. All rats were fed similar amounts of each diet so that there were no significant body weight differences between groups at the end of the 8 months. At periodic intervals all rats were injected with 1 micro c of [U-14C]glucose and placed in metabolism cages where a 24 hour urine sample was obtained. Urine was analyzed for sodium, sucrose and sucrose-14C content. Endogenous sucrose production was estimated from the percent of [U-14C]glucose recovered as urine sucrose-14C in 24 hours. All four hypotheses were confirmed.

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Pea leaves were illuminated in air containing 150 or 1000p.p.m. of 14CO2 for various times. Alternatively, segments of wheat leaves were supplied with [3-14C]serine for 40 min in the light in air with 145, 326 or 944p.p.m. of 12CO2. Sucrose was extracted from the leaf material, hydrolysed with invertase, and 14C in the pairs of carbon atoms C-3+C-4, C-2+C-5 and C-1+C-6 in the glucose moiety was measured. The results obtained after metabolism of 14CO2 were consistent with the operation of the photosynthetic carbon-reduction cycle; the effects of CO2 concentration on distribution of 14C in the carbon chain of glucose after metabolism of [3-14C]serine is more easily explained by metabolism through the glycollate pathway than by the carbon-reduction cycle.

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The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.

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