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  • Dictyostelium discoideum as a Model to Study Inositol Polyphosphates and Inorganic Polyphosphate.

Dictyostelium discoideum as a Model to Study Inositol Polyphosphates and Inorganic Polyphosphate.

Methods in molecular biology (Clifton, N.J.) (2019-11-28)
Yann Desfougères, Adolfo Saiardi
ABSTRACT

The yeast Saccharomyces cerevisiae has given us much information on the metabolism and function of inositol polyphosphates and inorganic polyphosphate. To expand our knowledge of the metabolic as well as functional connections between inositol polyphosphates and inorganic polyphosphate, we have refined and developed techniques to extract and analyze these molecules in a second eukaryotic experimental model, the amoeba Dictyostelium discoideum. This amoeba, possessing a well-defined developmental program, is ideal to study physiological changes in the levels of inositol polyphosphates and inorganic polyphosphate, since levels of both molecules increase at late stages of development. We detail here the methods used to extract inositol polyphosphates using perchloric acid and inorganic polyphosphate using acidic phenol. We also present the postextraction procedures to visualize and quantify these molecules by polyacrylamide gel electrophoresis and by malachite green assay.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
D-myo-Inositol 1,2,3,4,5,6-Hexakisphosphate, Dodecasodium Salt, Zea mays, Major phosphorus compound in plants that chelates with a variety of di- and trivalent cations.
Sigma-Aldrich
Orange G, for NA electrophoresis
Sigma-Aldrich
Phenol solution, Saturated with 0.1 M citrate buffer, pH 4.3 ± 0.2, BioReagent, for molecular biology
Sigma-Aldrich
N,N,N′,N′-Tetramethylethylenediamine, BioReagent, suitable for electrophoresis, ≥99.0%