Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Detergent solubilization and hydrophobic chromatography of rat brain phosphatidylinositol kinase

Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30–40 mM octylglucoside. Among these detergents, 1% Triton-X-100 was most effective in solubilizing the enzyme, and after treatment with, this agent, 100% of the activity was recovered in the high speed supernatant. Octylglucoside solubilized 40% of the enzyme at concentrations below its critical micelle concentration of 25 mM and up to 80% at higher levels. Solubilized phosphatidylinositol kinase failed to adsorb to adenosine nucleotide affinity resins. However, when the Triton-X-100 extract was chromatographed on an uncharged hydrophobic resin, consisting of dodecyl chains attached to Sepharose 4B by ether bonds, nearly all the enzyme activity was retained, and from 44–85% could be eluted with 8 mM sodium deoxycholate. Solubilization followed by hydrophobic chromatography resulted in several-fold purification of phosphatidylinositol kinase and may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.The abbreviations used are phosphatidylinositol 1,2-diacyl-sn-glycero-3-phosphoryl-1-l-myo-inositol - phosphatidylinositolphosphate 1,2-diacyl-sn-glycero-3-phosphoryl-1-l-myo-inositol-4-monophosphate - phosphatidylinositolbisphosphate 1,2-diacyl-sn-glycerol-3-phosphoryl-1-l-myo-inositol-4,5-bisphosphate - octylglucoside 1-0-n-octyl-d-glucopyranoside     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Pathways of d-fructose catabolism in species of Pseudomonas

Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P₂ were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P₂ aldolase, fructose-1,6-P₂ phosphatase, and P-hexose isomerase which could convert fructose-1,6-P₂ to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-¹⁴C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd ^- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd ^- mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

Rat L6 myotubes as an in vitro model system to study GLUT4-dependent glucose uptake stimulated by inositol derivatives

Some of inositol derivatives have been reported to help the action of insulin stimulating glucose uptake in skeletal muscle cells. Rat L6 myotubes were employed in an attempt to develop an in vitro model system for investigation of the possible insulin-like effect of eight inositol derivatives, namely allo-inositol, d-chiro-inositol l-chiro-inositol, epi-inositol, muco-inositol, myo-inositol, scyllo-inositol and d-pinitol. At a higher concentration of 1 mM seven inositol derivatives other than myo-inositol were able to stimulate glucose uptake, while at 0.1 mM only d-chiro-inositol, l-chiro-inositol, epi-inositol and muco-inositol could induce glucose uptake, indicating their significant insulin-mimetic activity. Immunoblot analyses revealed that at least d-chiro-inositol, l-chiro-inositol, epi-inositol, muco-inositol and d-pinitol were able to induce translocation of glucose transporter 4 (GLUT4) to plasma membrane not only in L6 myotubes but also in skeletal muscles of rats ex vivo. These results demonstrated that L6 myotubes appeared efficient as an in vitro system to identify inositol derivatives exerting an insulin-like effect on muscle cells depending on the induced translocation of GLUT4.     

The enzymes involved in the synthesis of phytic acid in Lemna gibba (Studies on the biosynthesis of cyclitols,XL.)

Summary The biosynthesis of phytic acid is known to be catalyzed by enzymes causing a stepwise phosphorylation of myo-inositol or 1l-myo-inositol 1-phosphate with adenosine triphosphate as phosphate donor. The kinases responsible for these phosphorylations in Lemna gibba were purified by affinity chromatography on a Sepharose gel carrying myo-inositol 2-phosphate at the binding site. Three fractions with enzymatic activity could be identified; in the first one, we find myo-inositol kinase (EC 2.7.1.64) phosphorylating myo-inositol to 1l-myo-inositol 1-phosphate; the second one brings about the phosphorylation of myo-inositol trisphosphate to phytic acid; the third one phosphorylates myo-inositol 1-phosphate to a myo-inositol trisphosphate. An enzyme oxidizing 1l-myo-inositol 1-phosphate to an uronic acid derivative is found in the first two fractions. In the presence of ATP, Mg²⁺ Mn²⁺, and the second and the third enzyme fractions in an appropriate mixture, 1l-myo-inositol 1-phosphate can be phosphorylated to phytic acid. The structure of the trisphosphate acting as an intermediate is not yet known.     

Phosphatidylinositol(4,5)bisphosphate and Phosphatidylinositol(4)phosphate in Plant Tissues

Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with [³H]myo-inositol or [³²P]Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as d-myo-inositol(1,4,5)trisphosphate and d-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.     

Uptake of auxotrophic cells of a heterocyst-forming cyanobacterium by tobacco protoplasts,and the fate of their associations

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 g per caryopsis or 1.5 mg g^–1 of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-³H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-³H]MI as well as d-[1¹⁴C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.Abbreviations MI myo-inositol - OCM-MI 2-O, C-methylene-myo-inositol     

Distribution of low molecular weight carbohydrates in the subgenusCeratotropis of the genusVigna (Leguminosae)

The seeds of 9 members of the subgenusCeratotropis (Piper) Verdc., namelyVigna aconitifolia (Jacq.) Maréchal,V. angularis (Willd.) Ohwi et Ohashi,V. minima (Roxb.) Ohwi et Ohashi,V. nakashimae (Ohwi) Ohwi et Ohashi,V. reflexo-pilosa Hayata,V. umbellata (Thumb.) Ohwi et Ohashi,V. mungo (L.) Hepper,V. radiata (L.) Wilczek andV. sp., have been examined. On their low molecular weight carbohydrate compositions, this subgenus has been divided into 2 subgroups, mungo-radiata group and angularis group. Four other species referred to the subgeneraPlectotropis (Schumach.) Bak.,Lasiospron (Benth. emend Piper) Maréchal, Mascherpa et Stainier andVigna, V. vexillata (L.) A. Rich.,V. lasiocarpa (Benth.) Verdc.,V. marina (Burm.) Merr. andV. unguiculata (L.) Walp., were also analyzed and they had distinctive carbohydrate compositions. 1d-4-O-methyl-myo-inositol and 1d-5-O-(α-d-galactopyranosyl)-4-O-methyl-myo-inositol were detected in all species examined exceptV. vexillata, V. mungo andV. radiata.     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

Role of inositol trisphosphate as a second messenger in signal transduction processes: An essay

This essay attempts to summarize some of the best evidence for the role of inositol trisphosphate as a second messenger in signal transduction processes. The following aspects are addressed in the essay: (a) The synthesis of inositol trisphosphate and other inositol lipids, (b) Receptor-phosphatidylinositol bisphosphate phospholipase C coupling and the N-ras protooncogene, (c) Inositol trisphosphate and intracellular calcium, (d) Cell growth and oncogenes, (e) Receptors linked to the phosphatidylinositol cycle, (f) Phototransduction and (g) Interactions between inositol trisphosphate and other second messengers.Abbreviations Cyclic AMP Adenosine 3,5-cyclic monophosphate - Cyclic GMP Guanosine 3,5-cyclic monophosphate - DG sn, 1,2-Diacylglycerol - EGF Epidermal growth factor - GDP Guanosine diphosphate - GTP Guanosine triphosphate - IP Inositol 1-monophosphate - IP₂ Inositol 1,4-diphosphate - IP₃ Inositol 1,4,5-trisphosphate - PA Phosphatidic acid - PDGF Platelet-derived growth factor - PI Phosphatidylinositol - PIP Phosphatidylinositol 4-monophosphate - PIP₂ Phosphatidylinositol 4,5-bisphosphate - PIP₃ Phosphatidylinositol 3,4,5-trisphosphate - PLC Phospholipase C     

High concentrations of d-pinitol or d-chiro-inositol inhibit the biosynthesis of raffinose family oligosaccharides in maturing smooth tare (Vicia tetrasperma [L.] Schreb.) seeds

In the present study we have investigated the effect of exogenous cyclitols on accumulation of their galactosides and raffinose family oligosaccharides (RFOs) in maturing smooth tare (Vicia tetrasperma [L.] Schreb) seeds. Feeding d-pinitol to pods of smooth tare increased the amount of free d-pinitol and its galactosides: galactopinitol A, galactopinitol B, di- and trigalactopinitol A in seeds. Similarly, feeding d-chiro-inositol, which does not occur naturally in Vicia seeds, resulted in the transport of this cyclitol in the seed, and caused accumulation of high levels of d-chiro-inositol galactosides (fagopyritol B1, B2 and B3). Accumulation of both cyclitols and their galactosides drastically reduced accumulation of verbascose and, to a lesser extent, stachyose and di-galactosyl- myo-inositol. Feeding d-chiro-inositol also decreased accumulation of di- and tri-galactosyl pinitols, naturally occurring in seeds. Inhibition of RFOs accumulation by elevated levels of free cyclitols indicates competition between biosynthesis of both types galactosides, and similarity of both biosynthetic pathways in smooth tare seeds.     

Potentiometric and P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP₆ ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP₆ aggregates. ³¹P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T₁ relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP₆ molecule results in an unstable heterogeneous system.     

Polyphosphoinositide phospholipase C and evidence for inositol-phosphate-hydrolysing activities in the plasma-membrane fraction from light-grown wheat (Triticum aestivum L.) leaves

The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca²⁺ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca²⁺ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP₂) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca²⁺ the enzyme clearly favoured PIP. In addition, the PIP₂-PLC activity was increased by Mg²⁺ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P₃ was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP₂-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC deoxycholate - IDPase inosine 5-diphosphatase - InsP_s inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e. - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PLC phospholipase C This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance.     

Pathways of d-fructose and d-glucose catabolism in marine species of Alcaligenes,Pseudomonas marina,and Alteromonas communis

Cell-free extracts of d-fructose grown cells of marine species of Alcaligenes as well as Pseudomonas marina contained an activity which catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose in the 1-position as well as activities of the following enzymes: 1-P-fructokinase, fructose-1,6-P₂ aldolase, PPi-dependent 6-P-fructokinase, fructokinase, glucokinase, P-hexose isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrase, and 2-keto-3-deoxy-6-P-gluconate aldolase. The presence of these enzyme activities would allow d-fructose to be degraded by the Embden-Meyerhof pathway and/or the Entner-Doudoroff pathway. In cell-free extracts of d-glucose grown cells, the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose as well as 1-P-fructokinase activity were reduced or absent while the remaining enzymes were present at levels similar to those found in d-fructose grown cells. Radiolabeling experiments suggested that both d-fructose and d-glucose were utilized primarily via the Entner-Doudoroff pathway. Alteromonas communis, a marine species lacking 1-P-fructokinase and the PPi-dependent 6-P-fructokinase, contained all the enzyme activities necessary for the catabolism of d-fructose and d-glucose by the Entner-Doudoroff pathway; the involvement of this pathway was also consistent with the results of the radiolabeling experiments.Non-Standard Abbreviations EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTS PEP: d-fructose phosphotransferase system - PPi-6-PFK PPi dependent 6-PFK - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

d-Mannitol dehydrogenase and d-mannitol-1-phosphate dehydrogenase in Platymonas subcordiformis: some characteristics and their role in osmotic adaptation

d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD⁺. The K _m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K _m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K _m values of the d-mannitol dehydrogenase were shifted to higher values. The K _m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P d-fructose 6-phosphate - Mes 2-(N-morpholino)ethanesulfonic acid - MT-DH d-mannitol-dehydrogenase - MT1P-DH d-mannitol-1-phosphate dehydrogenase - Pipes 1,4-piperazinediethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

sll1722, an unassigned open reading frame of<Emphasis Type="Italic"> Synechocystis</Emphasis> PCC 6803, codes for <Emphasis Type="SmallCaps">l</Emphasis>-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate synthase

l-myo-Inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes conversion of glucose 6-phosphate to l-myo-inositol 1-phosphate, the first and the rate-limiting step in the production of inositol, and has been reported from evolutionarily diverse organisms. Two forms of the enzyme have been characterized from higher plants, viz. cytosolic and chloroplastic, and the presence of MIPS has been earlier reported from the cyanobacteria (e.g. Spirulina sp.), the presumed chloroplast progenitors. The present study demonstrates possible multiple forms of MIPS and identifies the gene for one of them in the cyanobacterium Synechocystis sp. PCC 6803. Following detection of at least two immunologically cross-reactive MIPS forms, we have been able to identify from the fully sequenced Synechocystis genome an as yet unassigned open reading frame (ORF), sll1722, coding for the approx. 50-kDa MIPS protein, by using biochemical, molecular and bioinformatics tools. The DNA fragment corresponding to sll1722 was PCR-amplified and functional identity of the gene was confirmed by a complementation assay in Saccharomyces cerevisiae mutants containing a disrupted INO1 gene for the yeast MIPS. The sll1722 PCR product was cloned in Escherichia coli expression vector pET20b and the isopropyl -d-thiogalactopyranoside (IPTG)-induced overexpressed protein product was characterized following complete purification. Comparison of the sll1722 sequences with other MIPS sequences and its phylogenetic analysis revealed that the Synechocystis MIPS gene is quite divergent from the others.Abbreviations CBB Coomassie Brilliant Blue - EST Expressed sequence tag - G6P d-Glucose 6-phosphate - IPTG Isopropyl -d-thiogalactopyranoside - MIPS l–myo-Inositol 1-phosphate synthase - ORF Open reading frame     

Partial Purification and Characterization of Indol-3-Ylacetylglucosemyo-Inositol Indol-3-Ylacetyltransferase (Indoleacetic Acid-Inositol Synthase)

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-β-d-glucose to myo-inositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein. The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-d-galactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-β-d-glucose is, K_m = 30 micromolar, and for myo-inositol is, K_m = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.     

Cellular compartmentation of photosynthetic and photorespiratory enzymes in the heterocystous cyanobacterium Anabaena cylindrica

Activities of enzymes of photosynthesis and photorespiration have been measured in extracts of vegetative cells and heterocysts from the filamentous cyanobacterium Anabaena cylindrica. Phosphoribulokinase, d-ribulose 1,5-bisphosphate carboxylase/oxygenase, phosphoglycollate phosphatase and glycollate dehydrogenase activities were readily measured in vegetative cell extracts, but were undetectable or negligible in heterocyst preparations. The data help to explain why heterocysts are unable to perform photosynthetic CO₂ fixation. They also exemplify the co-ordinate compartmentation of enzymes of photosynthesis and photorespiration which occur in a differentiated phototrophic prokaryote.Abbreviations Ru5P d-ribulose 5-phosphate - RuBP d-ribulose 1,5-bisphosphate - DCPIP 2,6-dichlorophenolindophenol - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate