Determination of inositol 1,4,5-trisphosphate levels in Dictyostelium by isotope dilution assay.
A commercial isotope dilution assay was used for the determination of Ins(1,4,5)P3 levels in the microorganism Dictyostelium discoideum. Cross-reactivity in the assay was detected with extracts from cells and the medium. The compound which induced this cross-reactivity was tentatively identified as Ins(1,4,5)P3 by (i) codegradation with authentic [32P]Ins(1,4,5)P3 by three specific Ins(1,4,5)P3 phosphatases, and (ii) co-chromatography with authentic [32P]Ins(1,4,5)P3 on HPLC columns. The cellular concentration was estimated as 165 +/- 42 pmol/10(8) cells, yielding a mean intracellular Ins(1,4,5)P3 concentration of 3.3 microM. Dictyostelium cells secrete large amounts of Ins(1,4,5)P3 at a rate of about 10% of the cellular content per minute, yielding about 0.13 microM extracellular Ins(1,4,5)P3 after 15 min in a suspension of 10(8) cells/ml. The chemoattractant cAMP induced a transient increase of the Ins(1,4,5)P3 concentration; the data suggest an intracacellular rise from 3.3 to 5.5 microM with a maximum at 6 s after stimulation.
Van Haastert
P J
PJ
Cell Biology and Genetics Unit, University of Leiden, The Netherlands.
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000032:analysis* / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000032:analysis* / Q000096:biosynthesis; D011865:Radioisotope Dilution Technique; D013403:Sugar Phosphates / Q000032:analysis*
D007295:Inositol Phosphates; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP