We have reexamined the structure of inositol phosphates present in trophozoites of the parasitic amoeba Entamoeba histolytica and show here that, rather than being myo-inositol derivatives (Martin, J.-B., BakkerGrunwald, T., and Klein, G. (1993) Eur. J. Biochem. 214, 711-718), these compounds belong to a new class of inositol phosphates in which the cyclitol isomer is neoinositol. The structures of neo-inositol hexakisphosphate, 2-diphospho-neo-inositol pentakisphosphate, and 2,5-bisdiphospho-neo-inositol tetrakisphosphate, which are present in E. histolytica at concentrations of 0.08 -0.36 mM, were solved by two-dimensional 31 P-1 H NMR spectroscopy. No evidence for the co-existence of their myo-inositol counterparts has been found. These neoinositol compounds were not substrates of 6-diphosphoinositol pentakisphosphate 5-kinase, an enzyme purified from Dictyostelium discoideum that phosphorylates 6-diphospho-myo-inositol pentakisphosphate and more slowly also myo-inositol hexakisphosphate, specifically on position 5. Because preliminary data indicate that large amounts of the same neo-inositol phosphate and diphosphate esters are also present in another primitive amoeba, Phreatamoeba balamuthi, the occurrence of high concentrations of neo-inositol polyphosphates may be much more general than previously thought.Inositol phosphates are members of a large family of naturally occurring compounds that because of their complex role in cell signaling and homoeostasis have been intensively studied during the last two decades (1). Research has focused mainly on the commonly known myo-derivatives, although many other naturally occurring inositol stereoisomers are known. The most abundant and ubiquitous member of the family, myo-InsP 6 , has been shown to reach an intracellular concentration close to 1 mM in Dictyostelium discoideum (2, 3). Recently, new members of this class of compounds have been identified in cellular slime molds as 5-PP-and 6-PP-myo-InsP 5 1 and 3,5-bis-PP-and 5,6-bis-PP-myo-InsP 4 (4 -6). The same or similar highly phosphorylated and diphosphorylated compounds have been detected in free living and parasitic amoebae and a number of mammalian cell types (7-11). The latter molecules may function in cell signaling (2,(12)(13)(14). Entamoeba histolytica is a human intestinal parasite that causes amoebiasis. Its trophozoites contain high amounts of inositol polyphosphates (8). myo-Inositol is the major cyclitol building natural inositol polyphosphates, and other isomers are only rarely found and in very low concentrations. Consequently, the two major inositol phosphates of E. histolytica have been preliminarily identified as myo-inositol 2,4,6-triphosphate and 5-PP-myo-InsP 5 (8). However, we subsequently noticed during comparative studies by high resolution anion exchange chromatography that the inositol polyphosphates from E. histolytica were not eluted exactly with the same retention time as the myo-inositols used as reference. This casted some doubt on the previous identification. Therefore, we have r...

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Perchloric-acid extracts of axenic Entamoeba histolytica were investigated by 31P-NMR spectroscopy. All major 31P resonances observed were assigned to specific compounds. The cells contained inorganic phosphate (1039 nmol/g wet cells), pyrophosphate (16 nmol/g wet cells), nucleoside diphosphates (91 nmol/g wet cells), nucleoside triphosphates (275 nmol/g wet cells), NAD(P) (60 nmol/g wet cells), phosphocholine (1 84 nmol/g wet cells), phosphoethanolamine (214 nmol/g wet cells), cytidine 5'-diphosphocholine (41 nmollg wet cells) and cytidine 5'-diphosphoethanolamine (55 nmollg wet cells). The latter four compounds may act as intermediates in the salvage pathway for the synthesis of phosphatidylethanolamine and phosphatidylcholine. E. histolytica trophozoites also contained two inositol phosphates in large quantities, InsP, (0.26 ymol/g wet cells) and InsP, (0.11 ymollg wet cells). These components were identified by 31P-NMR, using homonuclear J-resolved and two-dimensional 1H-31P correlative, analyses as myo-inositol trisphosphate, Ins(2,4,6)P3, and pentakisphospho-myo-inositol diphosphate, Ins(1 ,2,3,4,6)Ps(5)P2.Entamoeba histolytica is the etiological agent of human amoebiasis. Its life cycle encompasses a trophozoite stage (a mobile, metabolically active form) and a cyst stage (a quiescent, infective form; Martinez-Palomo, 1982). In addition to being a parasite, E. histolytica is one of the most primitive eukaryotic cells known. It lacks mitochondria, peroxisomes and well-developed Golgi stacks and many of its biochemical features are more reminiscent of bacteria than of higher eukaryotic cells (reviewed in Bakker-Grunwald and Woestmann, 1993). The development of a method to grow trophozoites axenically (Diamond, 1968) has led to a rapid accumulation of information on the metabolism, membrane structure and membrane dynamics of these cells (reviewed in Martinez-Palomo, 1982;Reeves, 1984;Bakker-Grunwald, 1991).3'P-NMR spectroscopy is a valuable technique for studying cellular phosphate metabolism (Lundberg et al., 1990;Szwergold, 1992). The possibility of growing large amounts of E. histolytica trophozoites prompted us to use "P-NMR spectroscopy as an analytical tool to investigate their phosphorylated metabolites. We report here that E. histolytica contains high amounts of two inositol phosphates, Ins(2,4,6)- P , (InsP,) and Ins(1 ,2,3,4,6)P5(5)P, (InsP,). In addition to Pi, PPi, nucleoside diphosphates, nucleoside triphosphates and NAD(P), major metabolites were phosphoethanolamine, phosphocholine, CDPEtn and CDPCho. MATERIALS AND METHODSGrowth of E. histolytica amoebae E. histolytica HM1: IMSS (American type culture collection number 30459) was grown axenically at 36°C in TYI-S (trypticase/yeast-extracthrodserum) medium (Diamond et al., 1978) supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin. To harvest the cells, late-logphase cultures were chilled and centrifuged at 400Xg. The amoebae were then washed twice and resuspended in 180 mM NaC1, 10 mM Tris/HCl, pH 7.0. Perchloric-acid extracts of E...

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