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At least 757 records · Page 42Linked to original sources
PubMed1996

Evidence for the shikimate-3-phosphate interacting site in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis.

The role of basic amino acid residues that are highly conserved in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPs) in the binding of the substrate, shikimate-3-phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site-directed mutagenesis. Native and the mutant proteins were expressed using a two-vector system and the expressed proteins were purified to near homogeniety. The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity. The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N-terminal domain (K19 and R24) results in reduced affinity for shikimate-3-phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2006

Effects of sphingosine-1-phosphate and ceramide-1-phosphate on rat intestinal smooth muscle cells: implications for postoperative ileus.

Postoperative ileus, a major cause of morbidity after abdominal surgery, is characterized by intestinal dysmotility and inflammation. The aim was to investigate the involvement of sphingolipids in postoperative intestinal inflammation using a standardized rat model of intestinal surgical manipulation. Sphingolipid analysis (ESI-MS) of intestinal muscularis after manipulation revealed a time-dependent increase of sphingosine 1-phosphate (S1P) and of ceramide 1-phosphate (C1P). We therefore established a culture system of primary rat intestinal smooth muscle cells and examined the potential role of these sphingolipids in intestinal inflammation. Incubation of cells with either of the two sphingolipid-phosphates resulted in an elevated production of PGE(2). Further analysis revealed that S1P enhances cyclooxygenase 2 (COX-2) expression whereas C1P increases release of arachidonic acid, indicating an enhanced phospholipase A(2) activity. S1P-induced COX-2 expression was pertussis toxin sensitive, suggesting the involvement of Gi/o protein-coupled S1P receptors. Further downstream mediators of S1P induced COX-2 expression appear to be extracellular regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Collectively, our results demonstrate that intestinal smooth muscle cells represent a major target for both C1P and S1P activity. Thus, the sustained elevated concentration of the two bioactive sphingolipids in this tissue could at least in part explain postoperative intestinal dysmotility.

Animals↗
PubMed1997

Physical and Kinetic Evidence for an Association between Sucrose-Phosphate Synthase and Sucrose-Phosphate Phosphatase.

The possible formation of a multienzyme complex between sucrose (Suc)-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP) was examined by measuring the rates of Suc-6-phosphate (Suc-6-P) synthesis and hydrolysis in mixing experiments with partially purified enzymes from spinach (Spinacia oleracea) and rice (Oryza sativa) leaves. The addition of SPP to SPS stimulated the rate of Suc-6-P synthesis. SPS inhibited the hydrolysis of exogenous Suc-6-P by SPP when added in the absence of its substrate (i.e. UDP-glucose) but stimulated SPP activity when the SPS substrates were present and used to generate Suc-6-P directly in the reaction. Results from isotope-dilution experiments suggest that Suc-6-P was channeled between SPS and SPP. A portion of the SPS activity comigrated with SPP during native polyacrylamide gel electrophoresis, providing physical evidence for an enzyme-enzyme interaction. Taken together, these results strongly suggest that SPS and SPP associate to form a multienzyme complex.

Journal Article↗
PubMed1982

Effects of the relative extrachloroplastic concentrations of inorganic phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate on the rate of starch synthesis in isolated spinach chloroplasts.

The effect of external inorganic phosphate (Pi) on starch synthesis in isolated spinach (Spinacia oleracea American Hybrid No. 424) chloroplasts in the presence of millimolar concentrations of 3-phosphoglycerate (PGA) and/or dihydroxyacetone phosphate (DAP) was examined. Whereas CO(2) fixation was relatively constant as the ratio of the external phosphate to the PGA + DAP varied from 1:3 to 3:1, starch synthesis varied from 17% to 2% of the CO(2) fixation rate. With DAP alone, maximal starch synthesis was about 10% of the CO(2) fixation rate. The data demonstrate that the Pi/(PGA + DAP) ratio in the cytoplasm of plant cells could serve to regulate the flow of newly fixed carbon into starch without alterations in the rate of CO(2) fixation.

Journal Article↗
PubMed1982

Enantiomeric Form of myo-Inositol-1-Phosphate Produced by myo-Inositol-1-Phosphate Synthase and myo-Inositol Kinase in Higher Plants.

The product of myo-inositol-1-phosphate synthase, EC 5.5.1.4, from mature pollen of Lilium longiflorum Thunb., cv Ace (Easter lily) and that of myo-inositol kinase, EC 2.7.1.64, from wheat germ has been identified as 1l-myo-inositol-1-phosphate by gas chromatography of its trimethylsilyl-methyl phosphate derivative on a glass capillary column bearing a chiral phase.

Journal Article↗
PubMed1988

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase.

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 x 10(-7) molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45 degrees C, and the K(m) values for the substrates are 2.7 x 10(-6) molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 x 10(-6) molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.

Journal Article↗
PubMed1989

Species and Environmental Variations in the Effect of Inorganic Phosphate on Sucrose-Phosphate Synthase Activity : Reliability of Assays Based Upon UDP Formation.

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.

Journal Article↗
PubMed1992

Phosphate Deficiency in Maize : III. Changes in Enzyme Activities during the Course of Phosphate Deprivation.

The effects of low concentrations of phosphate (low-P) on soluble protein content, the activities of 12 different enzymes, and the rates of photosynthesis and respiration on the basis of leaf area were investigated in maize (Zea mays L.) leaves 16 to 24 days after planting (DAP). With low-P treatment, a drastic decrease in the rate of photosynthesis to only 6% of the maximum rate in control plants was observed by 24 DAP. Low-P treatment had almost no effect on the rate of respiration until 21 DAP, but then the rate of respiration decreased progressively to about 55% of the maximum rate in control plants. The soluble protein content in low-P plants decreased to 56% of the maximum content in control plants. The changes in the activities of enzymes in low-P plants showed several different patterns. The activities of pyruvate, orthophosphate dikinase, 3-phosphoglycerate kinase, phosphoenolpyruvate carboxylase (PEPC), ribulose 1,5-bisphosphate carboxylase, fructose 1,6-bisphosphate aldolase, catalase, phosphohexose isomerase, chloroplastic fructose 1,6-bisphosphatase, and ADP-glucose-pyrophosphorylase decreased steadily from 85 to 100% of the maximum activity found in 18- to 21-day-old control plants (V(max)) to 30 to 70% of V(max). The activity of sucrose phosphate synthase remained virtually constant at approximately 85 to 100% of V(max). The activity of UDP-glucose-pyrophosphorylase remained almost constant up to 21 DAP and then decreased to 80% of V(max) by 24 DAP. The activity of cytochrome c oxidase increased slightly up to 21 DAP but then decreased to 50% of V(max) by 24 DAP. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins, the subunit of PEPC stained less intensely in 24-d-old low-P plants. The possibility is discussed that during low-P treatment there is selective degradation of PEPC without a concomitant degradation of sucrose phosphate synthase, both of which are known to be localized in the cytoplasmic compartment of mesophyll cells.

Journal Article↗
PubMed1987

Nucleotide sequence and transcriptional startpoint of the glpT gene of Escherichia coli: extensive sequence homology of the glycerol-3-phosphate transport protein with components of the hexose-6-phosphate transport system.

The nucleotide sequences of the glpT gene of Escherichia coli and its regulatory region have been elucidated and the primary structure of the glycerol-3-phosphate transport protein deduced. Extensive amino acid sequence homology was found with two other cytoplasmic membrane proteins: the functionally related hexose-6-phosphate transport protein, and the UHPC protein involved in regulating hexose-6-phosphate uptake. Although no significant amino acid sequence homology was found with other transport proteins, such as the arabinose, citrate, glucose, melibiose, lactose or xylose transporters, all of these proteins share a common secondary structure arrangement with the GLP T protein as they apparently contain twelve membrane-spanning alpha-helical segments. The promoter for glpT was located by transcript mapping and shown to overlap a site to which catabolite activator protein binds in vitro. These findings indicate how catabolite repression may be mediated but do not explain its physiological significance in glycerol metabolism.

Amino Acid Sequence↗
PubMed2000

Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli.

To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K(m) values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k(cat) values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k(cat)/K(m)). The K(m) and k(cat) values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-beta-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

Catalysis↗
PubMed2004

Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum.

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.

Corynebacterium↗
PubMed1976

Modified 5'-nucleotides resistant to 5'-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl) uridine 5'-phosphate and N2, N2-dimethylguanosine 5'-phosphate from snake venom hydrolysates of transfer RNA.

A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents of RNA has recently been developed in this laboratory (Gray, M.W. Can. J. Biochem. 53, 735-746 (1975)). This assay method is based on the resistance of O2'-methylnucleoside 5'-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5'-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5'-nucleotides, each displaying an unusual resistance to 5'-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2, N2-dimethylguanosine 5'-phosphate (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U). Because of their resistance to 5'-nucleotidase, pm2/2G and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA. Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean +/- SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 +/- 0.03 (n=5) and 0.14 +/- 0.02 (n=4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase. The complete absence of pm2/2G in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2, N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5'-nucleotidase is a necessary but not sufficient criterion for concluding that a 5'-nucleotide is O2'-methylated. When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A.G. & Enger, M.D. Biochim. Biophys. Acta 349, 61-77 (1974)), may not be present in wheat embryo ribosomal RNA.

Guanine Nucleotides↗
PubMed1990

Distinctive regulation of the functional linkage between the human cation-independent mannose 6-phosphate receptor and GTP-binding proteins by insulin-like growth factor II and mannose 6-phosphate.

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.

Animals↗
PubMed1983

[Analysis and the stability of codeine phosphate. 2: Stability of aqueous codeine phosphate solutions].

The degradation velocity of aqueous codeine phosphate solutions is of first order. The formation of the degradation products (isomerization products) depends upon temperature, light and pH value. At pH = 3.5, codeine phosphate solutions are relatively stable. Atmospheric oxygen exerts no effect. Of the complexing agents and antioxidants tested, only citric acid and thiourea seem to be capable of reducing the rate of degradation. Besides glass ampoules, plastic containers of low-density polyethylene proved to be suited for codeine phosphate solutions.

Antioxidants↗
PubMed1995

The cation-independent mannose-6-phosphate receptor binds to soluble GPI-linked proteins via mannose-6-phosphate.

The cation-independent mannose-6-phosphate/insulin-like growth factor II receptor has been observed to bind to soluble forms of glycosyl-phosphatidylinositol-linked molecules, one of mammalian origin (rat Thy-1) and two of protozoan origins. Of the two phosphate groups found on the soluble forms of the protozoan glycosyl-phosphatidylinositol-linked molecules: (i) the internal mannose-6-phosphate diester (which forms a part of the ethanolamine bridge) and (ii) the inositol-1,2 cyclic phosphate group (which arises after cleavage of the membrane associated form with phosphatidylinositol-specific phospholipase C), only the former appears to be recognized by the mannose-6-phosphate/insulin-like growth factor II receptor, as mild acid hydrolysis which destroys the latter has been observed not to affect the receptor binding site.

Animals↗
PubMed1994

Synthesis of sphingosine-1-phosphate and dihydrosphingosine-1-phosphate.

The syntheses of D-erythro-sphingosine-1-phosphate and DL-erythro-sphinganine-1-phosphate are described starting from the commercially available D-erythro-sphingosine and DL-erythro-sphinganine. The phosphate group is introduced via phosphoramidite chemistry using bis(2-cyanoethyl)-N,N-diisopropylamino-phosphoramidite as the monophosphorylating reagent. The procedure generates the phosphorylated sphingoid bases in three steps and 32-39% overall yields.

Amides↗
PubMed1996

Tumor 31P NMR pH measurements in vivo: a comparison of inorganic phosphate and intracellular 2-deoxyglucose-6-phosphate as pHnmr indicators in murine radiation-induced fibrosarcoma-1.

Uncertainty regarding the intracellular/extracellular distribution of inorganic phosphate (P(i)) in tumors has raised concerns that pH calculated from the tumor P(i) chemical shift may not accurately represent the intracellular pH (pHin). This issue was addressed in subcutaneously transplanted murine radiation induced fibrosarcoma-1 by directly comparing pH measured via P(i) with pH measured via the in situ generated intracellular xenometabolite 2-deoxyglucose-6-phosphate (2DG6P). In 131 comparative measurements employing eight tumor-bearing mice under both control and hyperglycemic conditions (the latter to extend the range of tumor pH examined), the pH as derived from either 2DG6P or P(i) showed only a small, but statistically significant, difference (0.07 +/- 0.11 SD; P = 0.0001). Scatter in the comparative analysis over the pH range examined (ca. 5.5-7.5) was not uniform. Above pH 6.6, 2DG6P indicated a pH lower than that of P(i) by 0.088 +/- 0.105 SD (n = 107, P = 0.0001); below pH 6.6, 2DG6P indicated a pH essentially identical to and not statistically different from that of P(i) (mean difference 0.003 +/- 0.128 SD (n = 24, P = 0.92)). Evidence is presented in support of this differential arising from a systematic measurement error due to peak overlap between 2DG6P and endogenous phosphomonoester species. These results support the use of P(i) as a tumor 31P NMR pHin indicator, at least in RIF-1 tumors under control and hyperglycemic conditions.

Animals↗
PubMed2001

Essential role of residue H49 for activity of Escherichia coli 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme catalyzing the first step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid Synthesis.

The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme. To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E. coli and yeast transketolase (TK). Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity.

Amino Acid Sequence↗