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.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP 6 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-InsP 4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP 4 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.Keywords: myo-inositol; diphospho-myo-inositol phosphates; Dictyostelium; Polysphondylium.Cellular slime molds like Dictyostelium and Polysphondylium are ancient amoebae that are able to form multicellular aggregates when their food supply is exhausted. The cells of these aggregates differentiate in spore and stalk cells and finally form fruiting bodies. Alternatively, some species can form microcysts as a resistant stage when exposed to increased osmolarity. Under suitable conditions, spores or microcysts can germinate and release solitary living amoebae [1].Diphosphoinositol phosphates were originally discovered in the Dictyostelium discoideum wild-type strain NC4 [2]. In that study, an inositol phosphate derivative was observed that eluted after InsP 6 from an anion-exchange column. It was tentatively suggested that this compound, called`InsP y ', is a`pyrophosphate form of InsP 6 ' [3]. Meanwhile, InsP y from the genus Dictyostelium has been analyzed by HPLC and NMR and was found to consist of three different diphosphoinositol phosphates, namely 5-PP-InsP 5 , 6-PP-InsP 5 , and 5,6-bis-PP-InsP 4 . 5-PP-InsP 5 and 6-PP-InsP 5 can not be completely separated by anion-exchange HPLC but can be distinguished enzymatically using a kinase that specifically phosphorylates 6-PP-InsP 5 but not 5-PP-InsP 5 in isolated PP-InsP 5 fractions. In the closely related genus Polysphondylium, an additional compound of this type has been detected and identified as 1,5-bis-PP-InsP 4 or its enantiomer 3,5-bis-PP-InsP 4 [4,5].The metabolism of highly phosphorylated inositol phosphates during the development of Dictyostelium has been investigated in a previous study by monitoring the incorporation of [ 3 H]inositol into these compounds [3]. It was found that InsP 6 incorporates [ 3 H]inositol continuously for 8 h after onset of starvation. In contrast, no incorporation into InsP y , which corresponds to the sum of diphosphoinositol phosphates, could be seen during the first 3 h of starvation. After this lag phase, InsP y was linearly labeled by [ 3 H]inosit...

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