Shears S B, Ali N, Craxton A, Bembenek M E
Inositol Lipid Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 1995 May 5;270(18):10489-97. doi: 10.1074/jbc.270.18.10489.
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.
利用新获得的3H和32P标记底物，通过高效液相色谱法阐明了双二磷酸肌醇四磷酸（PP-InsP4-PP）的合成和代谢途径。还通过以结构诊断方式使用两种纯化的磷酸酶来鉴定代谢产物：烟草“焦磷酸酶”（Shinshi, H., Miwa, M., Kato, K., Noguchi, M., Matsushima, T., and Sugimura, T. (1976) Biochemistry 15, 2185 - 2190）和大鼠肝脏多肌醇多磷酸磷酸酶（MIPP；Craxton, A., Ali, N., and Shears, S. B. (1995) Biochem. J. 305, 491 - 498）。尽管MIPP在大鼠肝脏匀浆或完整的AR4-2J细胞中均未代谢大量这些多磷酸，但双磷酸肌醇多磷酸被MIPP水解的证明为其底物特异性提供了新信息。在肝脏匀浆中，肌醇六磷酸（InsP6）首先被磷酸化为双磷酸肌醇五磷酸（PP-InsP5），然后再转化为PP-InsP4-PP。这些激酶反应可被磷酸酶逆转，从而建立了两个偶联的底物循环。这两个去磷酸化反应可能由具有不同位置特异性以及对F-抑制敏感性不同（对PP-InsP5和PP-InsP4-PP的IC50值分别为0.03 mM和1.4 mM）的不同磷酸酶进行。在[3H]肌醇标记的AR4-2J细胞中，PP-[3H]InsP5和PP-[3H]InsP4-PP的稳态水平分别为[3H]InsP6水平的2 - 3%和0.6%。用0.8 mM NaF处理细胞40分钟揭示了这些多磷酸的持续周转，这使[3H]InsP6水平降低了50%，使PP-[3H]InsP5水平增加了16倍，使PP-[3H]InsP4-PP水平增加了5倍。用10 mM NaF处理的细胞中PP-[3H]InsP5水平也大幅增加，但随后未观察到PP-[3H]InsP4-PP水平有显著变化；这两种化合物周转的调控可能存在重要差异。
