Previous structural analyses of diphosphoinositol polyphosphates in biological systems have relied largely on NMR analysis. For example, in Dictyostelium discoideum, diphosphoinositol pentakisphosphate was determined by NMR to be 4- and/or 6-PPInsP5, and the bisdiphosphoinositol tetrakisphosphate was found to be 4,5-bisPPInsP4 and/or 5,6-bisPPInsP4 [Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315, 715-720]. We now describe three recent technical developments to aid the analysis of these compounds, not just in Dictyostelium, but also in a wider range of biological systems: (i) improved resolution and sensitivity of detection of PPInsP5 isomers by microbore metal-dye-detection HPLC; (ii) the use of the enantiomerically specific properties of a rat hepatic diphosphatase; (iii) chemical synthesis of enantiomerically pure reference standards of all six possible PPInsP5 isomers. Thus we now demonstrate that the major PPInsP5 isomer in Dictyostelium is 6-PPInsP5. Similar findings obtained using the same synthetic standards have been published [Laussmann, Reddy, Reddy, Falck and Vogel (1997) Biochem. J. 322, 31-33]. In addition, we show that 10-25% of the Dictyostelium PPInsP5 pool is comprised of 5-PPInsP5. The biological significance of this new observation was reinforced by our demonstration that 5-PPInsP5 is the predominant PPInsP5 isomer in four different mammalian cell lines (FTC human thyroid cancer cells, Swiss 3T3 fibroblasts, Jurkat T-cells and Chinese hamster ovary cells). The fact that the cellular spectrum of diphosphoinositol polyphosphates varies across phylogenetic boundaries underscores the value of our technological developments for future determinations of the structures of this class of compounds in other systems.

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