| Assay Method Information | |
| | In Vitro Activity Assay |
| Description: | In vitro PLD activity is measured with an exogenous substrate assay as previously described (Brown, H. A. et al. (1993) Cell 75:1137-1144). Briefly, PLD activity was measured as the release of [methyl-3H]choline from [choline-methyl-3H]dipalmitoylphosphatidylcholine. PLD enzyme (PLD1=3 nM, or PLD2=15 nM) is reconstituted with phospholipid vesicle substrates. Lipid solutions were dried and resuspended, and small unilamellar vesicles were prepared by bath sonication. All assays were performed at 37° C. with agitation for 30 min. Reactions were stopped by addition of trichloroacetic acid and bovine serum albumin. Free [methyl-3H]choline was separated from precipitated lipids and proteins by centrifugation and analyzed by liquid scintillation counting. |
| Affinity data for this assay | |
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