Phosphatidylinositol synthesis by a mn-dependent exchange enzyme in castor bean endosperm

myo-Inositol is incorporated into phosphatidylinositol by an exchange reaction associated with the endoplasmic reticulum fraction isolated from post-germination castor bean endosperm. The reaction requires Mn²⁺, has a pH optimum of 8.0, an apparent K_m for myo-inositol of 26 micromolar, and is stimulated about 15-fold by certain cytidine derivatives. The cytidine derivatives appear to be converted to CMP, which may be the only active stimulator. These optimal exchange reaction conditions, both with and without CMP, differ from those for cytidine-5′ -diphosphodiglyceride: myo-inositol transferase (EC 2.7.8), so the exchange does not appear to be a reversal of the transferase. This conclusion is augmented by the low rates of CDP-diglyceride formation from cytidine derivatives when compared to the high rate of myo-inositol incorporation into phosphatidylinositol in the presence of the same cytidine derivatives and identical reaction conditions.     

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.     

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.     

Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase

CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn²⁺ for activity. Maximal activity was at 1.5 millimolar MnCl₂. A K_m of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP-dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride.     

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.     

Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride:inositol transferase.

A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn²⁺ but not by Mg²⁺, while that in the presence of CDP-DG was activated by either Mn²⁺ or Mg²⁺. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds.     

Role of exogenous inositol and phosphatidylinositol in glycosylphosphatidylinositol anchor synthesis of GP49 by Giardia lamblia

Although Giardia lamblia trophozoites are unable to carry out de novo phospholipid synthesis, they can assemble complex glycophospholipids from simple lipids and fatty acids acquired from the host. Previously, we have reported that G. lamblia synthesizes GP49, an invariant surface antigen with a glycosylphosphatidylinositol (GPI) anchor. It is therefore possible that myo-inositol (Ins), phosphatidylinositol (PI) and other GPI precursors are obtained from the dietary products of the human small intestine, where the trophozoites colonize. In this report, we have investigated the role of exogenous Ins and PI on GPI anchor synthesis by G. lamblia. The results demonstrate that [³H]Ins and PI internalized by trophozoites, metabolically transformed into GlcN(acyl)-PI and downstream GPI molecules. Further investigations suggest that G. lamblia expresses cytidine monophosphate (CMP)-dependent (Mg²⁺-stimulated) and independent (Mn²⁺-stimulated) inositol headgroup exchange enzymes, which are responsible for exchanging free Ins with cellular PI. We observed that 3-deoxy-3-fluoro-D-myo-inositol (3-F-Ins) and 1-deoxy-1-F-scyllo-Ins (1-F-scyllo-Ins), which are considered potent inhibitors of Mn²⁺-stimulated headgroup exchange enzyme, inhibited the incorporation of [³H]Ins into PI and GPI molecules significantly, suggesting that CMP-independent (Mn²⁺-stimulated) exchange enzyme may be important for these reactions. However, 3-F-Ins and 1-F-scyllo-Ins were not effective in blocking the incorporation of exogenously supplied [³H]PI into GPI glycolipids. Thus, it can be concluded that G. lamblia can use exogenously supplied [³H]PI and [³H]Ins to synthesize GPI glycolipids of GP49; while PI is directly incorporated into GPI molecules, free Ins is first converted into PI by headgroup exchange enzymes, and this newly formed PI participates in GPI anchor synthesis.     

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na⁺- or H⁺-linked myo-inositol transporters. While Na⁺-coupled myo-inositol transporters are found exclusively in the plasma membrane, H⁺-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H⁺-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.     

The distribution of phosphatidylinositol in microsomal membranes from rat liver after biosynthesis de novo. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-³H]inositol or ³²P-labelled phospholipid. The ³²P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [³²P]P_i. The ³H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-³H]inositol into phosphatidylinositol by either exchange in the presence of Mn²⁺ or biosynthesis de novo in the presence of CTP and Mg²⁺. 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-³H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-³H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [³²P]phosphatidylinositol indicate that phosphatidyl[2-³H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.     

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.     

Phosphatidylinositol:myo-Inositol Exchange Activity in Intact Nerve Endings: Substrate and Cofactor Dependence, Nucleotide Specificity, and Effect on Synaptosomal Handling of myo-Inositol

Micromolar concentrations of CMP produced a large increase in Mn2+-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 microM, and that for myo-inositol was 38 microM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP. Manganese did not affect the uptake of myo-inositol into the synaptosomal cytosolic fraction or myo-inositol levels. Determinations of myo-inositol specific activity showed that the Mn2+-enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo-inositol. This lack of an effect on intrasynaptosomal myo-inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+-dependent exchange activity, it is the myo-inositol in the incubation medium that is being directly incorporated into membrane-bound phosphatidyl-inositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP-diacylglycerol:inositol phosphatidyl transferase, this enzyme is likely to be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+-dependent exchange in the absence of added nucleotide.     

Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L

Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [¹⁴C]Indole-3-acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3-acetyl-myo-inositol is the first seed auxin precursor to be identified.     

Phytate metabolism in Petunia pollen

Phytic acid has been detected in the anthers of young flower buds of Petunia hybrida, the amount increasing slowly as the flower develops until anther dehydration, when there was a more rapid increase in phytic acid content. In mature pollen, the phytic acid content was found to be 2.0 % by weight, of which 90 % was water soluble, while free myo-inositol was a relatively low 0.06 % by weight. Breakdown of phytic acid was initiated soon after pollen germination began, and its degradation products, myo-inositol and inorganic phosphate, were rapidly mobilized for phospholipid and pectin biosynthesis. Both are in high demand during pollen tube elongation. Utilization of myo-[2-³H]inositol for phospholipid biosynthesis was about five times that for pectin synthesis during the first few hours of pollen germination. The label in the phospholipid was identified as the myo-inositol moiety of phosphaltidylinositol, while the pectin material contained predominantly labelled arabinose, with smaller amounts of label in galacturonic acid, glucose and xylose. A chase experiment showed that the myo-inositol moiety of phosphatidylinositol was subject to a relatively rapid turnover, while the label in pectin was not. Labelling germinating pollen with [³²P]orthophosphate gave label in phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine of the phospholipids. Phosphatidylinositol contained 30 % of this label initially, a proportion which declined to 10 % over longer periods of germination.     

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.     

Studies on avian erythrocyte metabolism: purification and properties of myo-inositol 1-phosphatase form chick erythrocytes

An enzyme capable of hydrolyzing myo-inositol 1-phosphate was identified and partially purified from the erythrocytes of 7-day chicks. It has an apparent molecular weight of approximately 60,000, is heat stable, and has a pH of optimal activity between 6.5 and 7.3. In most regards the kinetic properties are similar to the myo-inositol 1-phosphatases of rat testis, rat mammary gland, bovine brain, and of yeast. The enzyme has an absolute requirement for a divalent cation; Mg²⁺ gave the greatest activity, with an optimal concentration of 2.5 mm in the standard assay employed. Zn²⁺, Co²⁺, and Mn²⁺ supported activity to a lesser degree. Activity was inhibited by NaF, HgCl₂, and p-hydroxymercuribenzoate. myo-Inositol tetrakis (dihydrogen phosphate) and myo-inositol 1,3,4,5,6-pentakis (dihydrogen phosphate) were not substrates for this enzyme and inhibited the hydrolysis of myo-inositol 1-phosphate. Unlike other phosphatases for myo-inositol 1-phosphate, this enzyme cleaved myo-inositol 1-phosphate (K_m = 8.6 × 10^?5 m) and myo-inositol 2-phosphate (K_m = 2.86 × 10^?4 m) at approximately the same rates. It also hydrolyzed 2′-purine and pyrimidine ribonucleotides about as well as myo-inositol 1-phosphate, but was only 20–30% as active against the 3′-ribonucleotides and had scarcely any activity against the 5′-ribonucleotides. The amount of enzyme activity in erythrocytes of embryos, chicks, and mature chickens was the same (~29 μmol/ml rbc/h). The biological function of this enzyme in avian erythrocytes is unclear at this time. Other tissues containing this phosphatase also have an enzyme which synthesizes myo-inositol 1-phosphate from glucose 6-phosphate, but we have been unable to detect the presence of such an enzyme in avian erythrocytes.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

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.     

Simple synthesis of <Superscript>32</Superscript>P-labelled inositol hexakisphosphates for study of phosphate transformations

Background and aims

In many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with ³²P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates.

Methods

We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-³²P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates.

Results

³²P-labeled inositol phosphates were separated by anion exchange HPLC with phosphate eluents. Additional HPLC methods were developed to allow facile separation of myo-, neo-, 1D-chiro- and scyllo-inositol hexakisphosphate on acid gradients.

Conclusions

We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[³²P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[³²P]2,3,4,[³²P]5,6–hexakisphosphate and 1D-chiro-inositol [³²P]1,2,3,4,5,[³²P]6-hexakisphosphate. Additionally, we describe HPLC separations of all inositol hexakisphosphates yet identified in soils, using a collection of soil inositol phosphates described in the seminal historic studies of Cosgrove, Tate and coworkers. Our study will enable others to perform radiotracer experiments to analyze fluxes of phosphate to/from inositol hexakisphosphates in different soils.

     

Effect of nucleotides on the incorporation of myo-inositol into phosphatidylinositol in rat liver microsomes

In rat liver microsomes the incorporation of inositol in the presence of Mn2+ was stimulated by cytidine nucleotides, whereas it was inhibited by other nucleotides. At low concentrations of CMP, AMP and other nucleotides stimulated inositol incorporation. No such effect was observed when the concentration of CMP was 2 mM or higher. It was found that an appreciable hydrolysis of CMP to cytidine and inorganic phosphate occurred during incubation with microsomes in the presence of Mg2+ or Mn2+. AMP was hydrolysed at a comparable rate. The activatory effect of AMP and other nucleotides on the CMP-dependent incorporation of inositol could be ascribed to protecting CMP against hydrolysis.     

A Novel Biosynthetic Pathway of Archaetidyl-myo-inositol via Archaetidyl-myo-inositol Phosphate from CDP-archaeol and d-Glucose 6-Phosphate in Methanoarchaeon Methanothermobacter thermautotrophicus Cells

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[¹⁴C]inositol 1-phosphate, which was enzymatically prepared from [¹⁴C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.