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PubMed1982

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of Tris.

1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH. 2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint. 3. Individual constants of rate of a simple transphosphorylation mechanism have been determined. 4. At high Tris concentration (greater than 1.0 M) a slight competitive inhibition has been observed. 5. Inhibition in NH4+-NH3Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH2 group of Tris is responsible for inhibition. 6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.

Alkaline Phosphatase↗
PubMed1993

Differential scanning calorimetric study of 5-enolpyruvoyl shikimate-3-phosphate synthase and its complexes with shikimate-3-phosphate and glyphosate: irreversible thermal transitions.

The thermal denaturation of native Escherichia coli 5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase, its binary complex with shikimate-3-phosphate (S3P) and its ternary complex with S3P and glyphosate have been studied using highly-sensitive differential scanning calorimetry (DSC). All observed transitions are strongly scanning-rate-dependent and irreversible. Consistent with these observations, the data were better fit by a simple irreversible model than by the controversial reversible model more commonly employed. The results obtained provide additional support for the application of irreversible models to the thermal denaturation of proteins. The calculated parameters, activation energy (Ea), enthalpy of denaturation (delta H) and transition temperature (Tm), obtained from fitting to an irreversible model agree well with values obtained from approximation techniques. Further, the results show that the formation of the ternary complex greatly enhances the thermal stability of the enzyme (delta Tm = 10.6 degrees C), while the binding of S3P alone increases the transition temperature only slightly (delta Tm = 3 degrees C). The heat of binding calculated at the transition temperature also demonstrates the greater stability of the ternary complex (delta H = -70 kcal/mol) versus the binary complex (delta H = -10 kcal/mol).

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Aldehyde-Lyases↗
PubMed2005

Enhancement of intracellular sphingosine-1-phosphate production by inositol 1,4,5-trisphosphate-evoked calcium mobilisation in HEK-293 cells: endogenous sphingosine-1-phosphate as a modulator of the calcium response.

Sphingosine-1-phosphate (S1P) regulates many cellular functions, such as migration, differentiation and growth. The effects of S1P are thought to be primarily mediated by G-protein coupled receptors, but an intracellular function as a calcium releasing second messenger has also been proposed. Here we show that in HEK-293 cells, exogenous S1P mobilises sequestered calcium by a mechanism primarily dependent on the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) pathway, and secondarily on the subsequent synthesis of intracellular S1P. Stimulating HEK-293 cells exogenously with S1P increased the production of both inositol phosphates and intracellular S1P. The calcium response was inhibited in cells treated with 2-APB, caffeine or U73122, showing that the PLC/IP3 pathway for calcium release is activated in response to exogenous S1P. The calcium response was partially inhibited in cells treated with the sphingosine kinase inhibitor DMS and in cells expressing a catalytically inactive sphingosine kinase, showing that endogenously produced S1P is also involved. Importantly, 2-APB and U73122 inhibited the S1P-evoked production of intracellular S1P. S1P is therefore not likely a major calcium releasing second messenger in HEK-293 cells, but rather a secondary regulator of calcium mobilisation.

Calcium↗
PubMed2005

Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol-1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase.

A reproducible approach to improve salt tolerance of conifers has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of three genotypes of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harboring the plasmid pBIGM which carrying two bacterial genes encoding the mannitol-1-phosphate dehydrogenase (Mt1D, EC 1.1.1.17) and glucitol-6-phosphate dehydrogenase (GutD) (EC 1.1.1.140), respectively. Transgenic plantlets were produced on selection medium containing 15 mg l(-1) kanamycin and confirmed by polymerase chain reaction (PCR) and Southern blot analysis of genomic DNA. The Mt1D and GutD genes were expressed and translated into functional enzymes that resulted in the synthesis and accumulation of mannitol and glucitol in transgenic plants. Salt tolerance assays demonstrated that transgenic plantlets producing mannitol and glucitol had an increased ability to tolerate high salinity. These results suggested that an efficient A. tumefaciens-mediated transformation protocol for stable integration of bacterial Mt1D and GutD genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

Agrobacterium tumefaciens↗
PubMed2005

Investigation of cytotoxic, genotoxic, mutagenic, and estrogenic effects of the flame retardants tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloropropyl)-phosphate (TCPP) in vitro.

The organophosphorus esters tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloropropyl)-phosphate (TCPP) have been widely used as flame retardants and fire preventing agents, e.g. in polyurethane foams. We investigated the cytotoxic, genotoxic, mutagenic, and estrogenic potentials of TCEP and TCPP, using different in vitro models. Cytotoxic effects were evaluated by neutral red uptake and genotoxicity with the alkaline single cell gel electrophoresis (Comet assay), both in V79 (hamster fibroblasts) cells. Mutagenicity was tested in the Ames assay with Salmonella typhimurium using the strains TA 97 a, 98, 100, 102, 104, 1535, 1537, and 1538, with and without metabolic activation by S9-rat liver homogenate. Estrogenic or anti-estrogenic effects were examined with the recombinant yeast reporter gene assay, and in human endometrial cancer Ishikawa cells by induction of alkaline phosphatase. In V79 cells TCEP was weakly cytotoxic at concentrations above 10 microM in the presence of S9-rat liver homogenate whereas TCPP showed cytotoxicity above 1mM in the presence of S9. Both substances did not induce DNA strand breaks in the alkaline version of the Comet assay neither without an external enzymatic metabolizing system, nor in the presence of S9-mix. Additionally, no mutagenic potential could be detected for TCEP and TCPP in eight Salmonella strains using concentrations up to 1mM in the presence and absence of S9. Hormonal activity shown as induction of estrogenic or anti-estrogenic effects could not be detected in the two in vitro test systems.

Animals↗
PubMedDate not supplied

Immunosuppressive activity of FTY720, sphingosine 1-phosphate receptor agonist: I. Prevention of allograft rejection in rats and dogs by FTY720 and FTY720-phosphate.

FTY720, a new class of immunomodulator, induces lymphopenia by sequestration of circulating lymphocytes into secondary lymphoid tissues. FTY720 at 0.1 to 1 mg/kg significantly prolonged the allograft survival in a dose-dependent manner and showed a marked synergistic effect in combination with cyclosporine (CsA) in rat skin and cardiac allograft models. In addition, the canine renal allograft survival was significantly prolonged by combination therapy with FTY720 at 0.03 to 1 mg/kg and CsA at 10 mg/kg as compared with monotherapy of FTY720 or CsA. By contrast, the combination therapy with CsA and azathioprine or CsA and mycophenolate mofetil resulted in only an additive effect in rat skin allograft. When FTY720 was administered to rats, FTY720 was metabolized by omega-oxidation of the octyl side chain, and beta-oxidation subsequently, or phosphorylated by sphingosine kinase. Omega- and beta-oxidized 4 metabolities of FTY720 at 10 mg/kg i.v. showed neither lymphopenia nor immunosuppressive activity in rat skin allograft. On the other hand, (S)-enantiomer of FTY720-phosphate at 0.1 and 1 mg/kg intravenously induced a marked lymphopenia and significantly prolonged the allograft survival in the rat allotransplantation. From these results, it is suggested the lymphopenia and the immunosuppression induced by FTY720 administration is due to the agonistic activity against SIP receptors of the active metabolite, (S)-FTY720-phosphate.

Animals↗
PubMed2003

Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection.

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.

Anions↗
PubMed1977

Synthesis of beta-L-fucopyranosyl phosphate and L-fucofuranosyl phosphates by the MacDonald procedure.

Fusion of beta-L-fucopyranose tetraacetate with phosphoric acid for 1 min at 50 degrees gives a 9:1 anomeric mixture of the alpha- and beta-pyranosyl phosphates. Longer fusion times give the alpha-anomer exclusively. The L-fucofuranose tetraacetates were synthesized for the first time by acetolysis of methyl-2,3,5-tri-O-acetyl-beta-L-fucofuranoside. Fusion of the furanose tetraacetates with phosphoric acid gave a mixture of the fucofuranosyl phosphates in which the beta-anomer predominated (beta/alpha= 2.4). Anomeric pairs in the fucofuranose series appear to be distinguishable by the chemical shift of the C-6 methyl protons, as already shown by Sinclair and Sleeter in the pyranose series.

Fucose↗
PubMed2001

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme-ligand complexes in solution, we have isolated the 26-kDa N-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate-3-phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N-terminal domain upon S3P binding.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2002

Sucrose metabolism: Anabaena sucrose-phosphate synthase and sucrose-phosphate phosphatase define minimal functional domains shuffled during evolution.

Based on the functional characterization of sucrose biosynthesis related protiens[SBP: sucrose-phosphate synthase (SPS), sucrose-phosphate phosphatase (SPP), and sucrose synthase (SuS)] in Anabaena sp. PCC7120 and sequence analysis, we have shown that SBP are restricted to cyanobacterium species and plants, and that they are multidomain proteins with modular architecture. Anabaena SPS, a minimal catalytic SPS unit, defines a glucosyltransferase domain present in all SPSs and SuSs. Similarly, Anabaena SPP defines a phosphohydrolase domain characteristic of all SPPs and some SPSs. Phylogenetic analysis points towards the evolution of modern cyanobacterial and plant SBP from a bidomainal common ancestral SPS-like gene.

Anabaena↗
PubMed1983

A comparison of estramustine phosphate versus cis-platinum alone versus estramustine phosphate plus cis-platinum in patients with advanced hormone refractory prostate cancer who had had extensive irradiation to the pelvis or lumbosacral area.

Single and combination chemotherapy was compared in a clinical trial for men with advanced, metastatic prostate cancer who had received prior pelvic irradiation and had had progression of disease despite hormonal therapy. The 149 patients were randomized to receive estramustine phosphate or cis-platinum alone or in combination. Of the 149 patients 25 (17 per cent) were excluded from the study but 124 were evaluated for response and survival. Entry variables were distributed similarly among patients in each treatment arm. There were no complete or partial responders but there were nearly twice as many patients whose disease was stabilized (33 per cent) on the combination regimen compared to estramustine phosphate (18 per cent) and about a third more than for cis-platinum (21 per cent). Analysis of survival revealed some advantage for patients on combination therapy. Major toxicities for all treatments were nausea and vomiting (62 to 88 per cent) and accompanying anorexia (72 to 95 per cent). Azotemia developed in 45 per cent of the patients receiving combination therapy. In addition an elevation in serum creatinine occurred in 22 per cent of the patients receiving combination therapy and in 17 per cent of those receiving cis-platinum alone. Myelosuppression occurred infrequently.

Aged↗
PubMed2002

Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway.

In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.

3T3 Cells↗
PubMed1999

A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzyme substrates for use with a screen-printed amperometric immunosensor for progesterone in cows' milk.

4-Aminophenyl phosphate (4-APP) and 1-naphthyl phosphate (1-NP) were compared as enzyme substrates for an amperometric milk progesterone biosensor utilising progesterone-conjugated alkaline phosphatase in a competitive immunoassay format. Cyclic voltammetry of the corresponding hydrolysis products, 4-aminophenol and 1-naphthol, at the surface of screen-printed carbon base transducers, uncoated or coated with anti-progesterone monoclonal antibody (mAb) showed well-defined anodic responses for both species, with the more sensitive being 4-aminophenol. Scan rate studies produced evidence that surface mAb could impede the diffusion of 4-aminophenol, but not 1-naphthol, toward the electrode surface. This was supported by computer simulation for the electrochemical rate constant (khet) using 4-aminophenol, which gave values at uncoated and mAb-coated electrodes of 6.5 x 10(-4) and 3.0 x 10(-4) cm s-1, respectively. The applied potential for oxidation of 4-aminophenol was 230 mV lower than for 1-naphthol. Nevertheless, by operating below +400 mV versus a saturated calomel reference electrode, it was possible to obtain a chronoamperometric signal for 1-naphthol in the absence of electrochemical interference from milk. Using mAb-coated SPCEs, calibration curves were obtained for progesterone in oestrus whole cow's milk spiked with standard concentrations over the range 0-50 ng/ml, using either 4-APP or 1NP as enzyme substrate. Precision values for triplicate sensors were 5.3-18.3% for 4-APP and 4.1-12.4% for 1-NP. An assay of real whole milk samples from different cows at various stages of the oestrus cycle produced correlations against a commercial EIA of r = 0.840 and 0.946 for 4-APP and 1-NP, respectively, 1-NP possesses the advantages over 4-APP of being inexpensive, easy to obtain and soluble (1-naphthol cf. 4-aminophenol) at high pH. From these observations, it is concluded that 1-NP is the preferred substrate for use with our proposed milk progesterone biosensor.

Alkaline Phosphatase↗
PubMed2003

Synthesis of fluorescence-labeled sphingosine and sphingosine 1-phosphate; effective tools for sphingosine and sphingosine 1-phosphate behavior.

A fluorescence-labeled sphingosine and sphingosine 1-phosphate have been successfully synthesized from the oxazolidinone methyl ester derived from glycidol via monoalkylation and the stereoselective reduction of the resulting ketone. The labeled sphingosine was converted into its phosphate by treatment with sphingosine kinase 1 (SPHK1) from mouse, and in platelets, and it was incorporated into the Chinese Hamster Ovarian (CHO) cells. In addition, MAPK was activated by NBD-Sph-1-P through Edg-1, Sph-1-P receptor.

4-Chloro-7-nitrobenzofurazan↗
PubMed2003

Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.

Amino Acid Sequence↗
PubMed2000

Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives.

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.

Amino Acid Sequence↗
PubMed2001

Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗