Synthesis and Metabolism of Bis-diphosphoinositol Tetrakisphosphate in Vitro and in Vivo

Abstract

The pathway of synthesis and metabolism of bis-diphosphoinositol tetrakisphosphate (PP-InsP4-PP) was elucidated by high performance liquid chromatography using newly available 3H- and 32P-labeled substrates. Metabolites were also identified by using two purified phosphatases in a structurally diagnostic manner: tobacco "pyrophosphatase" (Shinshi, H., Miwa, M., Kato, K., Noguchi, M. Matsushima, T., and Sugimura, T. (1976) Biochemistry 15, 2185-2190) and rat hepatic multiple inositol polyphosphate phosphatase (MIPP; Craxton, A., Ali, N., and Shears, S. B. (1995) Biochem. J. 305, 491-498). The demonstration that diphosphoinositol polyphosphates were hydrolyzed by MIPP provides new information on its substrate specificity, although MIPP did not metabolize significant amounts of these polyphosphates in either rat liver homogenates or intact AR4-2J cells. In liver homogenates, inositol hexakisphosphate (InsP6) was phosphorylated first to a diphosphoinositol pentakisphosphate (PP-InsP5) and then to PP-InsP4-PP. These kinase reactions were reversed by phosphatases, establishing two coupled substrate cycles. The two dephosphorylations were probably performed by distinct phosphatases that were distinguished by their separate positional specificities, and their different sensitivities to inhibition by F- (IC50 values of 0.03 mM and 1.4 mM against PP-InsP5 and PP-InsP4-PP, respectively). In [3H]inositol-labeled AR4-2J cells, the steady-state levels of PP-[3H]InsP5 and PP-[3H]InsP4-PP were, respectively, 2-3 and 0.6% of the level of [3H]InsP6. The ongoing turnover of these polyphosphates was revealed by treatment of cells with 0.8 mM NaF for 40 min, which reduced levels of [3H]InsP6 by 50%, increased the levels of PP-[3H]InsP5 16-fold, and increased levels of PP-[3H]InsP4-PP 5-fold. A large increase in levels of PP-[3H]InsP5 also occurred in cells treated with 10 mM NaF, but then no significant change to levels of PP-[3H]InsP4-PP were observed; there may be important differences in the control of the turnover of these two compounds.

The Structural and Functional Versatility of Inositol Phosphates

“…"InsP 7 "). A PP-InsP 5 isomer with the diphosphate group attached to the 5-carbon has, so far, been the only one found to occur in a variety of mammalian cell types (134 (133,179,182,183). The InsP 6 kinase has a value for ATP of 1.4 mM, which is sufficiently close to physiological levels so that changes in cellular [ATP] could impact on the concentration of PP-InsP 5 (182).…”

“…By blocking the dephosphorylation of PP-InsP 5 and [PP] 2 -InsP 4 in intact cells and exposing their ongoing rate of synthesis, F caused the levels of these compounds to increase manyfold within minutes. This application of F as a metabolic trap has shown that 30-50% of the cell's InsP 6 pool cycles through these diphosphorylated inositides every hour (133,134,183). The discovery of this effect (133) swept away another dogma of inositide metabolism that was prevalent in the 1980s: the idea that InsP 6 was metabolically lethargic (96).…”

“…The InsP 6 kinase has a value for ATP of 1.4 mM, which is sufficiently close to physiological levels so that changes in cellular [ATP] could impact on the concentration of PP-InsP 5 (182). (133,183). The diphosphorylated inositides appear to have arisen at an early evolutionary stage: 5-PP-InsP 5 has also been identified in Entomoeba histolytica (184) and Phreatamoeba balamuthi (185).…”

“…Nevertheless, it is now clear that there is considerable ongoing metabolic flux through PP-InsP 5 (133,183). By blocking the dephosphorylation of PP-InsP 5 and [PP] 2 -InsP 4 in intact cells and exposing their ongoing rate of synthesis, F caused the levels of these compounds to increase manyfold within minutes.…”

“…An (133,134,179,183,186). Estimates of the total cellular concentration of InsP 6 range from 5 to 100 μΜ (130)(131)(132).…”