title: Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis.
abstract: The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn't' play a major role in the IP₈ developmental increase.
authors.affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, United Kingdom ; Department of Biology, Ecology and Earth Science, University of Calabria, Rende, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Pisani
authors.affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.
authors.forename: Thomas
authors.initials: T
authors.lastname: Livermore
authors.affiliation: Department of Biology, Ecology and Earth Science, University of Calabria, Rende, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Rose
authors.affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.
authors.forename: Jonathan Robert
authors.initials: JR
authors.lastname: Chubb
authors.affiliation: Laboratory of Proteomics and Mass Spectrometry, Department of Experimental and Clinical Medicine, "Magna Græcia" University of Catanzaro, Catanzaro, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Gaspari
authors.affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.
authors.forename: Adolfo
authors.initials: A
authors.lastname: Saiardi
mesh_terms: D010832:6-Phytase / Q000494:pharmacology; D000143:Acids / Q000494:pharmacology; D002457:Cell Extracts; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D048788:Growth and Development / Q000187:drug effects; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry
chemical_list: D000143:Acids; D002457:Cell Extracts; D007295:Inositol Phosphates; D000242:Cyclic AMP; D010832:6-Phytase