Determination of inositol 1,4,5-trisphosphate levels in Dictyostelium by isotope dilution assay |
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Authors: | P J Van Haastert |
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Institution: | Cell Biology and Genetics Unit, University of Leiden, The Netherlands. |
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Abstract: | 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. |
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