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     

In <Emphasis Type="Italic">silico</Emphasis> study on the substrate binding manner in human <Emphasis Type="Italic">myo</Emphasis>-inositol monophosphatase 2

The human IMPA2 gene encoding myo-inositol monophosphatase 2 is highly implicated with bipolar disorder but the substrates and the reaction mechanism of myo-inositol monophosphatase 2 have not been well elucidated.9 In the present study, we constructed 3D models of three- and two-Mg²⁺-ion bound myo-inositol monophosphatase 2, and studied substrate-binding manners using the docking program AutoDock3. The subsequent study showed that the three-metal-ion model could interact with myo-inositol monophosphates, as follows: The phosphate moiety coordinated three Mg²⁺ ions, and the inositol ring formed hydrogen bonds with the amino acids conserved in the family. Furthermore, the OH group vicinal to the phosphate group formed a hydrogen bond with a non-bridging oxygen atom of the phosphate. These interactions have been proposed as crucial for forming the transitional state, bipyramidal structure in the bovine myo-inositol monophosphatase. We therefore propose that the human myo-inositol monophosphatase 2 interacts with myo-inositol monophosphates in the three-metal-ion bound form, and proceeds the dephosphorylation through the three-metal-ion theory.     

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.     

The Effect of ATP (Disodium Salt) upon Rotational Streaming

Through the continuous treatment with various solutions of ATP disodium salt the rotational streaming of the cytoplasma in barley root hairs has been stimulated about 1.2–1.7 times. With the concentrations employed the stimulation of the streaming did not depend on the external ATP supply, but on the initial rate of streaming. It is assumed that the main source of energy supporting the protoplasmic streaming is ATP. Therefore, the results obtained may be interpreted on the basis of variations in ATP content and its degradation products. The differences between initial rates of streaming are supposed to be due to variations of the endogenous ATP level. The ATP taken up probably stimulates the rotational streaming both through the supply of delivered energy and by lowering the cytoplasm viscosity. On the contrary, products of ATP hydrolysis increase the cytoplasm viscosity and induce a lowering or even cessation of the streaming.     

Myo-inositol oxygenase from oat seedlings

Summary Enzyme preparations from oat seedlings showing the activity ofmyo-inositol oxygenase (E.C.1.13.99.1) have been described previously. In contrast tomyo-inositol oxygenase preparations from other sources, e.g. rat kidney or yeast, the oat enzyme seemed to exhibit a somewhat less stringent activity, acting on other inositols and inositol methyl ethers as well as onmyo-inositol.By purification of the enzyme present in the extract from oat seedlings with the help of an affinity gel specific for enzymes acting onmyo-inositol a homogeneous enzyme preparation was obtained, which shows the same strict specificity as themyo-inositol oxygenase from other sources. It has a molecular weight of 62,000 and tends to aggregate to oligomers (up to tetramers) under physiological pH-values; in more alkaline media dissociation to monomers is observed. The action on the other inositols and inositol methyl ethers is apparently due to one or more other enzymes, which are also adsorbed on the affinity gel, but can be separated from themyo-inositol oxygenase by elution with increasing concentrations ofmyo-inositol.Dedicated to Professor Karl KRATZL on the occasion of his 60th birthday.     

Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate–fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.     

Potential role and therapeutic interests of myo-inositol in metabolic diseases

Several inositol isomers and in particular myo-inositol (MI) and D-chiro-inositol (DCI), were shown to possess insulin-mimetic properties and to be efficient in lowering post-prandial blood glucose. In addition, abnormalities in inositol metabolism are associated with insulin resistance and with long term microvascular complications of diabetes, supporting a role of inositol or its derivatives in glucose metabolism. The aim of this review is to focus on the potential benefits of a dietary supplement of myo-inositol, by far the most common inositol isomer in foodstuffs, in human disorders associated with insulin resistance (polycystic ovary syndrome, gestational diabetes mellitus or metabolic syndrome) or in prevention or treatment of some diabetic complications (neuropathy, nephropathy, cataract). The relevance of such a nutritional strategy will be discussed for each context on the basis of the clinical and/or animal studies. The dietary sources of myo-inositol and its metabolism from its dietary uptake to its renal excretion will be also covered in this review. Finally, the actual insights into inositol insulin-sensitizing effects will be addressed and in particular the possible role of inositol glycans as insulin second messengers.     

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.     

Phosphatidyl inositol: myo-Inositol exchange enzyme from rat liver: Partial purification and characterization

The enzyme which catalyzes CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol was solubilized from rat liver microsomes by sodium cholate and was partially purified by ammonium sulfate fractionation and sucrose density gradient centrifugation. Addition of phospholipids during purification and assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation contained about 3.7% of the protein and 35% of the original activity of the microsomal fraction. The activity of the enzyme preparation was strongly enhanced by addition of phosphatidyl inositol. The enzyme required Mn²⁺ for activity. The K_m for myo-inositol was 4 × 10^?5m. The pH optimum was 7.4. The activity was inhibited by thiol-reactive reagents and also to some extent by inosose-2 but not by scyllitol. Phosphorus-containing acidic substances such as acidic phospholipids and nucleotides were generally inhibitory. It was found that the preparation catalyzed liberation of inositol moiety from phosphatidyl inositol in a manner dependent on the concentration of free myo-inositol and also on Mn². The K_m of this reaction for free myo-inositol was estimated to be 7 × 10^?5m. This result indicates that CDP-diglyceride-independent incorporation, which has been assumed to show inositol exchange reaction, actually represents an exchange reaction between the myo-inositol moiety of phosphatidyl inositol and free myo-inositol. Phosphatidyl choline and phosphatidyl ethanolamine did not play a role as acceptor of the exchange reaction.     

Unimpaired formation of hormone-stimulated inositol triphosphate in human mesangial cells under hyperglycemic conditions

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-biphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grwon under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (K_m values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-triphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol triphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

Distribution and properties of CDP-diglyceride:inositol transferase from brain

CDP-diglyceride is converted to phosphatidyl inositol by several particulate subcellular fractions of guinea pig brain, with highest specific activity in the microsomal fraction. Optimal conditions with respect to pH, metal ion concentration, and substrate concentrations have been determined. The reaction was stimulated by the addition of bovine serum albumin and by Tween 80. Of several dl -CDP-diglycerides synthesized and used as substrates in a spectrophoto-metric assay for the enzyme, dl -CDP-didecanoin was the most active. The enzyme showed a high selectivity for myo-inositol. Of a number of compounds tested, only scyllo-inosose and epi-inosose served as substrates. Three inositol isomers and three myo-inositol monophosphates inhibited the reaction slightly. The most potent inhibitor found was galactinol, a myo-inositol galactoside.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

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.     

Über die Wirkung von ADP und einigen Kationen auf die Rotationsströmung in den Wurzelhaaren der Gerste (Hordeum vulgare L.)

Zusammenfassung In getrennten Versuchen wurde die Wirkung von ADP, Ca⁺⁺, Mg⁺⁺, K⁺ und Cu⁺⁺ auf die Rotationsströmung in den Wurzelhaaren der Gerste (Hordetim vulgare L.) untersucht. Das in verschiedenen Konzentrationen fortdauernd verabreichte ADP bedingte eine Stimulation der Plasmaströmung. Die Beschleunigung der Rotationsströmung war der ADP-Konzentration gegenüber umgekehrt proportional (Abb. 3).Von den untersuchten Kationen hatte nur Ca⁺⁺ (1·10^–3 Mol) eine Stimulationswirkung. Diese Stimulationswirkung wird der Aktivierung eines Enzyms bzw. eines kontraktilen Proteins mit ATPase-Eigenschaften zugeschrieben.Die Rolle von ADP und einigen Kationen bei der Stimulation der Rotation wurde dann mit Hilfe einer gemischten Behandlung untersucht. Diese bestand in der gleichzeitigen Verabreichung von ADP (1·10^–6 Mol) und CaCl₂, MgCl₂, KCl (1 · 10^–3 Mol) oder CuCl₂ (1·10^–6 Mol). Es wurde festgestellt, daß Mg⁺⁺ und Ca⁺⁺ eine antagonistische Wirkung ausüben. Ca⁺⁺ hebt die durch ADP induzierte Stimulation auf und reduziert die Rotationsgeschwindigkeit plötzlich bis auf den Kontrollwert. Die Mg⁺⁺-Wirkung bewirkt, nach einer zeitweiligen Beibehaltung der Stimulation, ebenfalls eine Abnahme der Geschwindigkeit. K⁺ hat eine ähnliche Wirkung wie Ca⁺⁺. Cu⁺⁺ beeinträchtigt die ADP-induzierte Stimulation in geringem Maße.Die gleichzeitige Einwirkung von ADP und einigen Kationen erlaubt die Aufstellung folgender Hypothese. Die Rotationsstimulation erfolgt dank dem ATP, das auf Kosten des von außen absorbierten ADP in den Mitochondrien synthetisiert wird. Die zusätzliche ATP-Synthese kann durch gleichzeitige Ca⁺⁺-Behandlung unterbunden werden. NachHanson und Mitarb, sollen Ca⁺⁺ und ADP um ein phosphoryliertes Zwischenprodukt in Kompetition treten, so daß es zu einer Ansammlung von Ca⁺⁺ und P_a in der Zelle kommt. Andererseits könnte teilweise auch die aktive, energieverbrauchende Salzabsorption die Geschwindigkeitsabnahme der Rotation bei gemischter Behandlung erklären.

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The effect of ADP and some cations on rotational streaming in barley (Hordeum vulgare L.) root hairs

Summary The effect of ADP, Ca⁺⁺, Mg⁺⁺, K⁺, and Cu⁺⁺ upon rotational streaming within barley (Hordeum vulgäre L.) root hairs was separately studied. It was shown that various solutions of ADP may stimulate the streaming after continuous treatment. The rate increase of the rotational streaming was inverse proportional to ADP concentration (Fig. 3).From the investigated cations only Ca⁺⁺ (1·10^–3M) caused a stimulation of streaming after continuous treatment. This effect is probably due to enzymic activation of a contractile proteine which has ATPase feature.The role of ADP and of the investigated cations in the stimulation of the rotational streaming was studied by means of mixed treatment. This kind of treatment consists in a simultaneous administration of ADP (1 · 10^–6M) and CaCl₂, MgCl₂, KCl (1 · 10^–3M), or CuCl₂ (1 · 10^–6M) solutions. Ca⁺⁺ and Mg⁺⁺ showed an antagonistic action. Ca⁺⁺ brings about an immediately suppress of ADP induced stimulation. Suddenly the rate of streaming comes back to control. Mg⁺⁺ after a temporary maintaining of stimulation, also causes the lowering of the streaming. The action of K⁺ was very similar to those of Ca⁺⁺. Cu⁺⁺ changes to a little extent the stimulation caused by ADP.The simultaneous action of ADP and of the investigated cations allow us to express the following hypothesis. The stimulation of the rotational streaming after ADP treatment probably is due to ATP synthetized in mitochondria on the account of ADP. The additional synthesis of ATP can be prevented by simultaneous administration of Ca⁺⁺. According toHanson and his coworkers Ca⁺⁺ would compete with ADP for a phosphorylated intermediate product. From a such competition would result the Ca⁺⁺ and P_i accumulation. The active uptake of salts which require energy would also explain the lowering of the rotational streaming rate after the mixed treatment.

     

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.     

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.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase

Myo-inositol may be incorporated in the formation of phosphatidylinositol by two mechanisms. One reaction utilizes CDP-diacylglycerol and is catalyzed by phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11). The second reaction is the phosphatidylinositol: myo-inositol exchange reaction, in which a free inositol is exchanged for an existing inositol headgroup. This characterization of inositol incorporation into phosphatidylinositol in the green alga Chlamydomonas reinhardtii provides evidence for the presence of both reactions. The transferase reaction required a divalent cation and exhibited its maximum activity at 2.0 mM Mn²⁺. The optimal pH for this reaction was 8.5–9.0. The best substrate concentrations were 0.5 mM CDP-diacylglycerol and 1.2 mM myo-inositol, with an estimated K_m for myo-inositol of 0.2 mM. The exchange reaction also required Mn²⁺ for activity, but became saturated at 0.5 mM Mn²⁺. The optimal pH of the exchange reaction was 8.0, the optimal myo-inositol concentration was 0.3 mM, and the estimated K_m for myo-inositol in this reaction was 0.015 mM. Measurement of the transferase reaction in cell fractions of C. reinhardtii indicated that the activity occurred primarily in the microsomal fraction, with little or no activity in the plastids.     

Characteristics of a phosphatidylinositol exchange activity of soybean microsomes

Microsome fractions from hypocotyls of dark-grown soybean (Glycine max [L.] Merrill) seedlings incorporated myo-inositol into phosphatidylinositol by an exchange reaction stimulated by Mn²⁺ (optimum at 10 mm) and cytidine nucleotides (CMP = CDP CTP) but not by Mg²⁺ or nucleotides other than cytidine nucleotides. The activity was membrane associated, with an optimum pH of 8, stimulated by auxin, and inhibited by certain thiol reagents or by heating above 40°C. With radioactive inositol, phosphatidylinositol was the only radioactive product. That turnover was by myo-inositol exchange was verified from experiments where unlabeled inositol replaced already incorporated inositol with approximately the same kinetics as for the incorporation of label. Both the incorporation and the displacement reactions were stimulated by Mn²⁺ and CMP and both were responsive to auxin with comparable dose dependency. Corresponding exchange activities with choline or ethanolamine were not observed. The phosphatidylinositol-myo-inositol exchange activity was low or absent from plasma membrane, tonoplast, and mitochondria enriched fractions. The activity co-localized on free-flow electrophoresis and aqueous two-phase partition with NADPH cytochrome c reductase and latent IDPase, markers for endoplasmic reticulum and Golgi apparatus, respectively. With microsomes incubated with both ATP and inositol, polyphosphoinositides were unlabeled demonstrating separate locations for the inositol exchange and phosphatidylinositol kinase reactions. Thus, the auxin-responsive inositol turnover activity of soybean membranes is distinct from the usual de novo biosynthetic pathway. It is not the result of a traditional D-type phospholipase and appears not to involve plasma membrane-associated polyphosphoinositide metabolism. It most closely resembles previously described phosphatidylinositol-myo-inositol exchange activities of plant and animal endoplasmic reticulum.     

Defective Craniofacial Development and Brain Function in a Mouse Model for Depletion of Intracellular Inositol Synthesis

myo-Inositol is an essential biomolecule that is synthesized by myo-inositol monophosphatase (IMPase) from inositol monophosphate species. The enzymatic activity of IMPase is inhibited by lithium, a drug used for the treatment of mood swings seen in bipolar disorder. Therefore, myo-inositol is thought to have an important role in the mechanism of bipolar disorder, although the details remain elusive. We screened an ethyl nitrosourea mutant mouse library for IMPase gene (Impa) mutations and identified an Impa1 T95K missense mutation. The mutant protein possessed undetectable enzymatic activity. Homozygotes died perinatally, and E18.5 embryos exhibited striking developmental defects, including hypoplasia of the mandible and asymmetric fusion of ribs to the sternum. Perinatal lethality and morphological defects in homozygotes were rescued by dietary myo-inositol. Rescued homozygotes raised on normal drinking water after weaning exhibited a hyper-locomotive trait and prolonged circadian periods, as reported in rodents treated with lithium. Our mice should be advantageous, compared with those generated by the conventional gene knock-out strategy, because they carry minimal genomic damage, e.g. a point mutation. In conclusion, our results reveal critical roles for intracellular myo-inositol synthesis in craniofacial development and the maintenance of proper brain function. Furthermore, this mouse model for cellular inositol depletion could be beneficial for understanding the molecular mechanisms underlying the clinical effect of lithium and myo-inositol-mediated skeletal development.