Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.1.14 (SPS)
813 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for product analysis that eliminates a problematic step in the radiometric sucrose-phosphate synthase assay is described. The method uses chromatography on a boronate-derivatized high-performance liquid chromatography column to separate the labeled product, [14C]sucrose phosphate, from unreacted uridine 5'-diphosphate-[14C]glucose (UDP-Glc). Direct separation of these compounds eliminates the need for treatment of the reaction mixtures with alkaline phosphatase, thereby avoiding the problem of high background caused by contaminating phosphodiesterase activity in alkaline phosphatase preparations. The method presented in this paper can be applied to many UDP-Glc requiring enzymes; here we show its use for determining the activities of sucrose-phosphate synthase, sucrose synthase, and uridine diphosphate-glucose pyrophosphorylase in plant extracts.
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PMID:A high-performance liquid chromatography-based radiometric assay for sucrose-phosphate synthase and other UDP-glucose requiring enzymes. 183 Jul 27

The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-phosphate synthase (SPS) was identified by overexpressing part of the gene from spinach (Spinacia oleracea). Degenerate oligonucleotide primers corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polymerase chain reaction to isolate a partial cDNA clone. Comparison of the deduced amino acid sequence of spinach SPS with the sequences of potato sucrose synthase showed that the partial cDNA included one region that was highly conserved between the proteins. Expression of the partial cDNA clone of SPS in Escherichia coli produced a 26-kD fusion protein that photoaffinity labeled with 5-N3UDP-Glc. Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portion of the SPS protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide from the photolabeled protein provided tentative identification of amino acid residues that make up the uridine-binding domain of SPS.
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PMID:Identification of the uridine-binding domain of sucrose-phosphate synthase. Expression of a region of the protein that photoaffinity labels with 5-azidouridine diphosphate-glucose. 810 11

As measured 7, 14, and 21 days after the application of 10(-2) M vanadyl sulfate solution to the foliage of 4.5-month-old sugar beet plants, significantly less growth of the leaves and an increase in the sucrose content of the storage root resulted. Accompanying these alterations were a higher rate of carbon dioxide fixation, a lower rate of respiration, and a decreased rate of nitrate reductase, glutamic-pyruvic transaminase, phosphatase, and invertase activity. The enzymes of sucrose synthesis, sucrose synthetase, sucrose phosphate synthetase and uridine diphosphate glucose-pyrophosphorylase were stimulated. The content of reducing sugar, nitrite N, amino acids and protein was less, and that of nitrate N was greater in the vanadium-treated plants. In the majority of cases the greatest magnitude of change occurred during the first 7 days following treatment. The changes in growth and chemical composition are believed to be closely related to the stimulation or inhibition of the various enzymes by vanadyl sulfate.
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PMID:Effect of Vanadium on Growth, Chemical Composition, and Metabolic Processes of Mature Sugar Beet (Beta vulgaris L.) Plants. 1665 5

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by (14)CO(2) assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total (14)CO(2) fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = -0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.Four nodulated soybean cultivars were grown to maturity in a greenhouse. Fully expanded leaves at the top of the canopy were sampled during vegetative growth (45 days), at flowering (79 days), and at mid-podfill (120 days). In general, activities of SPS and uridine-5'-diphosphatase were highest during vegetative growth, and they decreased during reproductive development, whereas activity of sucrose synthase and leaf starch content tended to increase. Leaf starch was negatively correlated with levels of SPS (r = -0.71). The results support the postulate that sucrose-P synthetase is a key control point regulating the photosynthetic formation of sucrose, and, hence, starch.
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PMID:Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves. 1666 77

Levels of fructose 2,6-bisphosphate (F2,6BP) and related metabolites were measured in 8- or 9-day-old barley (Hordeum vulgare L.) primary leaves throughout a 24 hour cycle. Young barley leaves contained about 0.4 nanomole F2,6BP per milligram chlorophyll at the end of a 12 hour dark period. F2,6BP levels increased rapidly following a dark-to-light transition and then decreased to about 0.1 nanomole per milligram chlorophyll after 5 or 10 minutes of light. Low levels of F2,6BP were detected in barley primary leaves throughout the day. A 10-fold increase in F2,6BP was observed during the first hour of the dark period and then levels of this metabolite decreased slowly for the next several hours. Only small diurnal fluctuations were noted in barley leaf glucose 6-phosphate and uridine 5'-diphosphoglucose levels. There were rapid changes in whole leaf F2,6BP levels when the light intensity was altered. High F2,6BP levels in the dark were not observed after short photosynthetic periods. Results obtained with barley primary leaves support the suggestion that F2,6BP is involved in regulating the flow of photosynthate from the chloroplast to sucrose. Extractable sucrose-phosphate synthase activity was inversely related to barley primary leaf F2,6BP levels. This finding may indicate that the activities of sucrose-phosphate synthase and cytosolic fructose 1,6-bisphosphatase in barley primary leaves are metabolically coordinated.
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PMID:Control of photosynthetic sucrose synthesis in barley primary leaves: role of fructose 2,6-bisphosphate. 1666 83

The effect of inorganic phosphate (Pi) on sucrose-phosphate synthase (SPS) activity was determined for the enzyme from five plant species (Nicotiana tabacum L., Spinacia oleracea L., Triticum aestivum L., Zea mays L., Glycine max L.) using two assay methods. The assay method based on determination of uridine diphosphate glucose- (UDPG) and fructose-6-phosphate-dependent sucrose formation was linear up to 15 minutes for all species tested. When assayed in this way, the effect of Pi at levels of 5 or 10 millimolar in the assay was variable, ranging from 0 to 35% inhibition of SPS activity. The assay method based on substrate dependent UDP formation was linear for some, but not for all of the species tested. Deviations from linearity were caused by loss of UDP from the assay medium. In some species, the extent of UDP loss was influenced by the level of Pi in the assay medium and, for at least one species (tobacco), it was influenced by the environment in which the plants were grown. The results indicated that (a) the role of Pi as an effector of SPS may vary depending on the species, and (b) the UDP assay method should be used with caution for assays of crude or desalted extracts, particularly when evaluating the effect of Pi on SPS activity.
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PMID:Species and Environmental Variations in the Effect of Inorganic Phosphate on Sucrose-Phosphate Synthase Activity : Reliability of Assays Based Upon UDP Formation. 1666 54

The effect of low phosphate supply (low P) was determined on the diurnal changes in the rate of carbon export, and on the contents of starch, sucrose, glucose, and fructose 2,6-bisphosphate (F2,6BP) in leaves. Low-P effects on the activities of a number of enzymes involved in starch and sucrose metabolism were also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers and the low-P treatment induced nutritionally. Low-P treatment decreased carbon export from the leaf much more than it decreased photosynthesis. At growth chamber photon flux density, low P decreased carbon export by 34% in light; in darkness, export rates fell but more so in the control so that the average rate in darkness was higher in low-P leaves. Low P increased starch, sucrose, and glucose contents per leaf area, and decreased F2, 6BP. The total extractable activities of enzymes involved in starch and sucrose synthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5-diphosphoglucose pyrophosphorylase, and sucrose-phosphate synthase. The activities of some enzymes involved in starch and sucrose breakdown were also increased by low P. We propose that plants adapt to low-P environments by increasing the total activities of several phosphatases and by increasing the concentrations of phosphate-free carbon compounds at the expense of sugar phosphates, thereby conserving Pi. The partitioning of carbon among the various carbon pools in low-P adapted leaves appears to be determined in part by the relative capacities of the enzymes for starch and sucrose metabolism.
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PMID:Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet: III. Diurnal Changes in Carbon Partitioning and Carbon Export. 1666 61

We have investigated whether sucrose accumulation in heterotrophic cell-suspension cultures of Chenopodium rubrum L. is regulated by a cycle in which sucrose is simultaneously synthesised and degraded. Net sucrose accumulation was measured by monitoring the sucrose content, unidirectional synthesis was monitored by supplying pulses of [(14)C] glucose, and unidirectional degradation was estimated from the difference between unidirectional synthesis and net accumulation. When 50 mM glucose was supplied to carbohydrate-depleted cells there was a rapid net accumulation of sucrose, which stopped after 24 h. The incorporation of (14)C into sucrose was similar to the initial rate of net sucrose accumulation, but rapid (14)C incorporation continued after the cells had stopped accumulating sucrose. A method was developed to rapidly separate sucrose-phosphate synthase (SPS) from uridine-diphosphate-hydrolysing activities which interfered with the assay. The cells contained enough SPS activity to catalyse the observed rate of sucrose synthesis. SPS activity increased in cells which had stopped accumulating sucrose, and the enzyme became less sensitive to inhibition by inorganic phosphate. Sucrose synthase and alkaline invertase activity were four- and twofold higher than SPS activity, and both degradative enzymes increased in cells which had stopped accumulating sucrose. Sucrose synthase is strongly modulated by the concentration of sucrose and by competitive feedback regulation by fructose in these cells. It is concluded that sucrose accumulation ceases in these cells because the rate of degradation of sucrose increases until it matches the rate of synthesis. It is discussed how this cycle is regulated, and how it may interact with the substrate cycle between triose-phosphates and hexose-phosphates (Hatzfeld and Stitt, 1990, Planta 180, 198-204). These cycles allow sucrose turnover to respond in a highly sensitive manner to small changes in the balance between the supply of sucrose and the demand for carbon for respiration and biosynthesis in the cell.
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PMID:Cytosolic cycles regulate the turnover of sucrose in heterotrophic cell-suspension cultures of Chenopodium rubrum L. 2419

Sucrose phosphate synthetase (EC 2.4.1.14) is the key enzyme for sucrose synthesis in Dunaliella tertiolecta. It has been partially purified and characterized. The enzyme contains one binding site for uridine diphosphoglucose and two binding sites for fructose-6-phosphate; it is allosterically controlled by fructose-6-phosphate. Inorganic phosphate stimulates the enzymic activity, particularly in the presence of higher concentrations of fructose-6-phosphate. Sucrose phosphate synthetase is not halophilic or halotolerant. The temperature dependence of the enzymic activity cannot fully explain the observed increase in sucrose synthesis in Dunaliella by elevated temperature.
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PMID:Sucrose biosynthesis in Dunaliella : II. Isolation and properties of sucrose phosphate synthetase. 2441 71

Availability of plant-specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measured in vitro, often under non-physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximal in vivo catalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome-b6f complex, ATP-citrate synthase, sucrose-phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition-specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements from Arabidopsis thaliana rosette with and fluxes through canonical pathways in a constraint-based model of leaf metabolism. In comparison to findings in Escherichia coli, we demonstrate weaker concordance between the plant-specific in vitro and in vivo enzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximal in vivo catalytic rates, and available quantitative metabolomics data are below reported K M values and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome-wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation.
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PMID:Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana. 3265 14


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