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

(1) Partially purified preparations of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) contain an endogenous protein kinase that phosphorylates and inactivates the enzyme with [gamma-32P]ATP. (2) The kinetic effect of phosphorylation is to alter affinities for substrates and the effector inorganic phosphate without affecting maximum velocity. (3) Two-dimensional peptide mapping of tryptic digests of in vitro labeled SPS yielded two phosphopeptides (designated sites 5 and 7). Labeling of the two sites occurred equally with time, and both correlated with inactivation. Maximum inactivation was associated with incorporation of 1.5 to 2.0 mol P/mol SPS tetramer, and about 70% of the phosphoryl groups were incorporated into one of the sites (phosphopeptide 7). (4) Phosphorylation and inactivation were strongly inhibited by NaCl, and the presence of salt alters some characteristics of the kinase reaction. In the absence of salt, the apparent Km for Mg.ATP was estimated to be 5 microM. (5) The dependence of the rate of phosphorylation on SPS concentration suggested that SPS and the protein kinase are distinct enzymes, but have some tendency to associate especially in the presence of ethylene glycol. (6) Ca2+/EGTA and polyamines have no effect on the rate of phosphorylation, whereas polycations (polylysine, polybrene and protamine) are inhibitory. (7) Of the metabolic intermediates tested, Glc 6-P inhibited phosphorylation and inactivation of the enzyme. The inhibition was not antagonized by inorganic phosphate, which suggests that Glc 6-P may be an effector of the kinase, rather than the target protein. Regulation by Glc 6-P may be of physiological significance.
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PMID:In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase. 182 91

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 aim of this work was to use preparations from germinating seeds of Pisum sativum to determine the apparent equilibrium constant of the reaction catalysed by sucrose-phosphate synthase (EC 2.4.1.14) and to compare this with the mass-action ratio of the reaction in the seeds. The apparent equilibrium constant ranged from 5.3 at 0.25 mM-MgCl2, pH 7.0, to 62 at 10 mM-MgCl2, pH 7.5. The sucrose phosphate content of the seeds, 23 nmol/g fresh wt., was determined by separating sucrose phosphate from sucrose by ion-exchange chromatography and then measuring the sucrose released by alkaline phosphatase. Comparison of equilibrium constants and mass-action ratios in the cotyledons of 38 h-germinated seeds showed that the reactions catalysed by glucose-6-phosphate isomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase are close to equilibrium, and those catalysed by sucrose-phosphate synthase and sucrose phosphatase are considerably displaced from equilibrium in vivo.
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PMID:Apparent equilibrium constant and mass-action ratio for sucrose-phosphate synthase in seeds of Pisum sativum. 214 Feb 58

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) extracted from darkened spinach (Spinacia oleracea L.) leaves has a low activation state, defined as the ratio of activity measured with limiting substrates (plus the inhibitor Pi) to activity with saturating substrates (maximum velocity). Preincubation at 25 degrees C of desalted crude extracts from darkened leaves resulted in a time-dependent increase in activation state that was inhibited by Pi [IC50 (concentration causing 50% inhibition) approximately 3 mM], molybdate, okadaic acid (IC50 approximately 25 nM) and vanadate, but was stimulated by fluoride. The "spontaneous activation" of SPS in vitro was enhanced slightly by exogenous MgCl2 (up to 5 mM) and exhibited a pH optimum of 7.0 to 7.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32P]Pi in the dark was lost with time when extracts were incubated at 25 degrees C. This loss in radiolabel was substantially reduced by vanadate. These results provide direct evidence for action of an endogenous protein phosphatase(s) using SPS as substrate. The spontaneous activation achieved in vitro could be reversed by subsequent addition of 1 mM Mg.ATP; the activation/inactivation achieved in vitro was similar in magnitude to the dark-light regulation observed in vivo. Moreover, feeding okadaic acid to excised leaves in the dark blocked subsequent light activation of SPS without affecting photosynthetic rate. These results are consistent with the notion that SPS contains phosphorylation site(s) that reduce enzyme activation state and that dephosphorylation of these residue(s) is the mechanism of light activation. Regulation of the protein phosphatase by Pi may be of physiological significance.
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PMID:Activation of sucrose-phosphate synthase from darkened spinach leaves by an endogenous protein phosphatase. 217 86

Studies were conducted to determine whether protein phosphorylation may be a mechanism for regulation of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS), shown previously to be light-dark regulated by some type of covalent modification. Radioactive phosphate was incorporated into the 120-kDa subunit of SPS during labeling of excised leaves with [32P]Pi, as shown by immunoprecipitation and denaturing gel electrophoresis of the enzyme. Conditions which activated the enzyme (illumination of leaves or mannose treatment of leaf discs in darkness) reduced the incorporation of radiolabel into SPS in the in vivo system. The partially purified SPS protein could also be phosphorylated in vitro using [gamma-32P]ATP. In the in vitro system, the incorporation of radiolabel into the 120-kDa subunit of SPS was dependent on time and magnesium concentration, and was closely paralleled by inactivation of the enzyme. These results provide the first evidence to establish protein phosphorylation as a mechanism for the covalent regulation of SPS activity.
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PMID:Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity. 252 12

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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PMID:The effect of sucrose on the rate of de novo sucrose biosynthesis in leaf protoplasts from spinach, wheat and barley. 640 85

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated by reversible protein phosphorylation. When the enzyme is phosphorylated it is inactivated and can be reactivated by removal of phosphate. The major regulatory phosphorylation site is known to be Ser158 in the spinach-leaf enzyme, and two protein kinase activities have been resolved chromatographically which phosphorylate SPS at this site in vitro. In this report, we use a set of synthetic peptide substrate analogs based on the phosphorylation site sequence, and a set of Escherichia coli-expressed 26-kDa fragments of spinach SPS which contain the site, to identify the recognition elements that target the two protein kinases to Ser158. The major recognition element consists of basic residues at P-3 and P-6 relative to the phosphorylated serine. Comparison of the spinach enzyme amino-acid sequence with two other plant species show conservation of these amino acids and implies that these signals are also conserved. We also present evidence that glucose-6-phosphate is not only an allosteric activator of SPS but also an inhibitor of SPS-protein kinase per se, thereby allowing it to act at both levels of SPS regulation.
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PMID:Characterization of the substrate specificity of sucrose-phosphate synthase protein kinase. 763 38

The activity of a type 2A protein phosphatase from spinach leaves was monitored using phosphorylated sucrose-phosphate synthase (SPS) as a substrate. After partial purification the overall activities of sucrose-phosphate synthase phosphatase (SPS-P) recovered from leaves harvested in the dark and in the light did not vary. However, SPS-P preparations from darkened leaves were more strongly inhibited by inorganic phosphate and certain phosphorylated compounds than preparations from illuminated or mannose fed leaves. We conclude, that activation of SPS involves an interconversion of multiple forms of SPS-P activity.
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PMID:Sucrose-phosphate synthase phosphatase, a type 2A protein phosphatase, changes its sensitivity towards inhibition by inorganic phosphate in spinach leaves. 822 58

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.
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PMID:Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase. 827 10

The possible presence of a sucrose-phosphate synthase (SPS) activating/stabilizing factor (SAF) presumably lost during SPS purification was investigated. Rice leaf protein extracts were chromatographed in a DEAE-Sephacel column. SPS activity of previously purified rice enzyme was enhanced to different extent by aliquots of fractions from such column. The activating capacity could not be replaced by albumin, but was nullified by EDTA. When the fractions were boiled or treated with TCA, the activating capacity disappeared suggesting its proteinaceous nature. The presence of 10 microM okadaic acid had no effect on the stimulatory action of SAF on SPS denying the possibility to SAF to be a SPS-phosphatase. Although it overlaps somehow with sucrose synthase (SS) in DEAE-Sephacel fractions, the activating protein factor and SS eluted separately during Sephadex G-200 chromatography. The activating ability was saturable at a fixed SPS concentration and was able to enhance SPS activity from other plant sources. Simultaneous studies on the activities of SPS and sucrose-phosphate phosphatase (SPP), closely linked to SPS, allowed us to suggest that SAF could be SPP. The presence of SAF/SPP did not alter the affinity of SPS for its substrates but helped to reverse the Pi inhibition at low Fru-6-P concentrations. We conclude that SPS may possibly interact with SPP, contributing to a more effective sucrose synthesis.
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PMID:Activation of sucrose-phosphate synthase by a protein factor/sucrose-phosphate phosphatase. 883 97


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