Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C.
We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
Cubitt
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Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Firtel
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D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004023:Dictyostelium / Q000201:enzymology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*
D007295:Inositol Phosphates; D008563:Membrane Lipids; D010169:Palmitic Acids; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D015544:Inositol 1,4,5-Trisphosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D002118:Calcium