SYNTHESIS AND METABOLISM OF BIS-DIPHOSPHOINOSITOL TETRAKISPHOSPHATE IN-VITRO AND IN-VIVO

The pathway of synthesis and metabolism of bis-diphosphoinositol tetra kisphosphate (PP-InsP(4)-PP) was elucidated by high performance liquid chromatography using newly available H-3- and P-32-labeled substrates . Metabolites were also identified by using two purified phosphatases in a structurally diagnostic manner: tobacco ''pyrophosphatase'' (Shin shi, Ii,, Miwa, M., Kato, K., Noguchi, M. Matsushima, T., and Sugimura , T. (1976) Biochemistry 15, 2185-2190) and rat hepatic multiple inosi tol polyphosphate phosphatase (MIPP; Craxton, A., All, N., and Shears, S. B. (1995) Biochem. J. 305, 491-498). The demonstration that diphos phoinositol polyphosphates were hydrolyzed by MIPP provides new inform ation on its substrate specificity, although MIPP did not metabolize s ignificant amounts of these polyphosphates in either rat liver homogen ates or intact AR4-2J cells. In liver homogenates, inositol hexakispho sphate (InsP(6)) was phosphorylated first to a diphosphoinositol penta kisphosphate (PP-InsP(5)) and then to PP-InsP(4)-PP. These kinase reac tions were reversed by phosphatases, establishing two coupled substrat e cycles. The two dephosphorylations were probably performed by distin ct phosphatases that were distinguished by their separate positional s pecificities, and their different sensitivities to inhibition by F- (I C50 values of 0.03 mM and 1.4 mM against PP-InsP(5) and PP-InsP(5)-PP, respectively). In [H-3]inositol-labeled AR4-2J cells, the steady-stat e levels of PP-[H-3]InsP(5) and PP-[H-3]InsP(4)-PP were, respectively, 2-3 and 0.6% of the level of [H-3]InsP(6). The ongoing turnover of th ese polyphosphates was revealed by treatment of cells with 0.8 mM NaF for 40 min, which reduced levels of [H-3]InsP(6) by 50%, increased the levels of PP-[H-3]InsP(5) 16-fold, and increased levels of PP-[H-3]In sP(4)-PP 5-fold. A large increase in levels of PP-[H-3]InsP(5) also oc curred in cells treated with 10 mM NaF, but then no significant change to levels of PP-[H-3]InsP(4)-PP were observed; there may be important differences in the control of the turnover of these two compounds.