BIOLOGICAL VARIABILITY IN THE STRUCTURES OF DIPHOSPHOINOSITOL POLYPHOSPHATES IN DICTYOSTELIUM-DISCOIDEUM AND MAMMALIAN-CELLS

Previous structural analyses of diphosphoinositol polyphosphates in bi ological systems have relied largely on NMR analysis. For example, in Dictyostelium discoideum, diphosphoinositol pentakisphosphate was dete rmined by NMR to be 4- and/or 6-PPInsP(5), and the bisdiphosphoinosito l tetrakisphosphate was found to be 4,5-bisPPInsP(4) and/or 5,6-bisPPI nsP(4) [Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J . 315, 715-720]. We now describe three recent technical developments t o aid the analysis of these compounds, not just in Dictyostelium, but also in a wider range of biological systems: (i) improved resolution a nd sensitivity of detection of PPInsP(5) isomers by microbore metal-dy e-detection HPLC; (ii) the use of the enantiomerically specific proper ties of a rat hepatic diphosphatase; (iii) chemical synthesis of enant iomerically pure reference standards of all six possible PPInsP(5) iso mers. Thus we now demonstrate that the major PPInsP, isomer in Dictyos telium is 6-PPInsP(5). Similar findings obtained using the same synthe tic 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 Dictyosrelium PPInsP(5) pool is comprised of 5-PPInsP(5). The biological significance of this new observation was reinforced by our demonstration that 5-PPInsP, is the pre dominant PPInsP, isomer in fou r different mammalian cell lines (FTC human thyroid cancer cells, Swis s 3T3 fibroblasts, Jurkat T-cells and Chinese hamster ovary cells). Th e fact that the cellular spectrum of diphosphoinositol polyphosphates varies across phylogenetic boundaries underscores the value of our tec hnological developments for future determinations of the structures of this class of compounds in other systems.