Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

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).     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

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.     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

Osmotic regulation ofmyo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H₂O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H₂O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H₂O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 g/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Metabolism of myo-Inositol and growth in various sugars of suspension-cultured tobacco cells

Tobacco (Nicotiana tabacum L.) cells were cultured in a liquid medium which contained sucrose as a source of carbon and energy. Various cell-wall constituents and wall precursors (L-arabinose, D-xylose, D-galactose, D-mannose, D-glucuronate, myo-inositol) were added to cells growing in this medium to by-pass possible rate-limiting steps in the relevant metabolic pathways. None of these compounds stimulated growth as measured by increase in fresh weight; myo-inositol did cause a slight increase and L-arabinose a decrease in dry weight accumulation compared to controls grown on sucrose only. Although myo-inositol was not needed for rapid growth, tracer level amounts of [2-³H]myo-inositol were rapidly absorbed and metabolized. Label was incorporated into the uronide and pentose residues of cell walls and exocellular polysaccharide.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination

Radiolabeled d-[1-³H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the ³H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained ³H, showing that de novo synthesis of myo-inositol from [1-³H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The ³H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, ³H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.     

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.     

Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passage of carboxyfluorescein in staminal hairs ofSetcreasea purpurea

pH-buffered carboxyfluorescein (Buffered-CF) alone (control), or Buffered-CF solutions containing one of the following: (1)d-myo-inositol (I); (2)d-myo-inositol 2-monophosphate (IP₁); (3)d-myo-inositol 1,4-bisphosphate (IP₂); (4)d-myo-inositol 1,4,5-trisphosphate (IP₃); (5)d-fructose 2,6-diphosphate (F-2,6P₂) were microinjected into the terminal cells of staminal hairs ofSetcreasea purpurea Boom. Passage of the CF from this terminal cell along the chain of cells towards the filament was monitored for 5 min using fluorescence microscopy and quantified using computer-assisted fluorescence-intensity video analysis. Cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either I, IP₁ or F-2,6P₂ was similar to that in hairs microinjected with Buffered-CF only. On the other hand, cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either IP₂ or IP₃ was inhibited. These results indicate that polyphosphoinositols may be involved in the regulation of intercellular transport of low-molecular-weight, hydrophilic molecules in plants.Abbreviations CF 5(6)Carboxyfluorescein - DG diacylglycerol - F2, 6P₂ d-fructose 2,6-diphosphate - I d-myo-inositol - IP₁ d-myo-inositol 2-monophosphate - IP₂ d-myo-inositol 1,4-bisphosphate - IP₃ d-myo-inositol 1,4,5-trisphosphate     

Effect of Ammonia and Methionine Sulfoximine on myo-Inositol Transport in Cultured Astrocytes

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myo-inositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P lt; 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0 ± 0.5 nmol/hour/mg protein which was reduced to 7.45 ± 0.27 and 7.02 ± 0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5 ± 4.52, 44.4 ± 5.82, and 39.3 ± 7.0 M, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 M, respectively. Treatment of astrocytes with either 5 or 10 mM NH₄Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.     

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     

Identification of 3-hydroxy-6-methyl-2H-pyran-2-one from pyrolysis of phosphoric acid-treated cellulosic materials

The hydrogen isotope-effect that occurs in vitro during myo-inositol 1-phosphate synthase-catalyzed conversion of d-[5-³H]glucose 6-phosphate into myo-[2-³H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the ³H/¹⁴C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70 % ethanol-insoluble fraction of d-[5-³H, 1-¹⁴-C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of d-glucose that occurred during its conversion into d-galactosyluronic residues of pectic substance is not explained by loss of ³H when UDP-d-[5-³H, 1-¹⁴C]glucose is oxidized by UDP-d-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.     

Metabolism of myo-Inositol by Germinating Lilium longiflorum Pollen

Lilium Iongiflorum pollen tubes absorbed myo-[2-³H]inositol produced labeled metabolites which were separated into acid-soluble and -insoluble fractions. The soluble fraction contained labeled myo-inositol, d-glucuronic acid, myo-inositol 1-phosphate, and at least three other unidentified compounds. The acid-insoluble fraction contained considerable chloroformsoluble radioactivity and a labeled residue. Labeled myo-inositol was also absorbed by germinating pollen prior to the time of pollen tube initiation; however, there was a marked reduction in amounts of myo-inositol 1-phosphate and glucuronic acid produced by this pollen in comparison with growing pollen tubes.     

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.     

Asymmetric Distribution of Glucose and Indole-3-Acetyl-myo-Inositol in Geostimulated Zea mays Seedlings

Indole-3-acetyl-myo-inositol occurs in both the kernel and vegetative shoot of germinating Zea mays seedlings. The effect of a gravitational stimulus on the transport of [³H]-5-indole-3-acetyl-myo-inositol and [U-¹⁴C]-d-glucose from the kernel to the seedling shoot was studied. Both labeled glucose and labeled indole-3-acetyl-myo-inositol become asymmetrically distributed in the mesocotyl cortex of the shoot with more radioactivity occurring in the bottom half of a horizontally placed seedling. Asymmetric distribution of [³H]indole-3-acetic acid, derived from the applied [³H]indole-3-acetyl-myo-inositol, occurred more rapidly than distribution of total ³H-radioactivity. These findings demonstrate that the gravitational stimulus can induce an asymmetric distribution of substances being transported from kernel to shoot. They also indicate that, in addition to the transport asymmetry, gravity affects the steady state amount of indole-3-acetic acid derived from indole-3-acetyl-myo-inositol.     

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.     

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.