Diphospho-myo-inositol phosphates in Dictyostelium and Polysphondylium: identification of a new bisdiphospho-myo-inositol tetrakisphosphate.

The two major diphospho inositol phosphates from the axenic strain Dictyostelium discoideum AX2 were previously investigated and identified as 6-PP-InsP5 and 5,6-bis-PP-InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non-axenic wild-type species of Dictyostelium and two of Polysphondylium were studied. It was found that all of the Dictyostelium species exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondylium species contain 5-PP-InsP5 as the predominant isomer. Besides 5,6-bis-PP-InsP4, a new bis-PP-InsP4 was detected in Polysphondylium. This compound is either 1,5-bis-PP-InsP4 or its corresponding enantiomer 3,5-bis-PP-InsP5. The structures were elucidated by two-dimensional 1H-1H and 1H-31P NMR analysis. Additionally, they were confirmed using a specific 6-PP-InsP(5)-5-kinase from D. discoideum AX2 as an enantio-specific tool and enantiomerically pure reference standards.

Structures of diphospho-myo-inositol pentakisphosphate and bisdiphospho-myo-inositol tetrakisphosphate from Dictyostelium resolved by NMR analysis.

Diphospho-myo-inositol phosphates (PP-InsP5 and bis-PP-InsP4) were isolated from Dictyostelium in order to clarify the precise positional isomerism by two-dimensional 1H/31P-NMR analysis. The diphosphorylated inositol phosphates are 4-PP-Ins(1,2,3,5,6)P5 and 4,5-bis-PP-Ins(1,2,3,6)P4 or their corresponding enantiomers. The vicinal arrangement of the diphospho groups with its steric and electrostatic constraints possibly qualifies bis-PP-InsP4 as a metabolite with high phosphate-group-transfer potential in phosphotransferase reactions.

Diphospho-myo-inositol phosphates from Dictyostelium identified as D-6-diphospho-myo-inositol pentakisphosphate and D-5,6-bisdiphospho-myo-inositol tetrakisphosphate.

Two diphospho-myo-inositol phosphates from Dictyostelium were recently investigated by two-dimensional 1H/31P NMR analysis and assigned to be either D-4-diphospho-myo-inositol pentakisphosphate (D-4-PP-InsP5) and D-4,5-bisdiphospho-myo-inositol tetrakisphosphate (D-4,5-bis-PP-InsP4) or their corresponding enantiomers D-6-PP-InsP5 and D-5,6-bis-PP-InsP4. In the present study the naturally occurring enantiomers were identified by using defined synthetic PP-InsP5 isomers as substrates for a partially purified PP-InsP5 5-kinase from Dictyostelium. This enzyme specifically phosphorylates the naturally occurring PP-InsP5 and the synthetic D-6-PP-InsP5, leading to D-5,6-bis-PP-InsP4. In contrast, neither D-4-PP-InsP5 nor D-1-PP-InsP5 or D-3-PP-InsP5 are converted by the enzyme.

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.

neo-inositol polyphosphates in the amoeba Entamoeba histolytica.

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., Bakker-Grunwald, 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 neo-inositol. 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 neo-inositol compounds were not substrates of 6-diphospho-inositol 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.