Figure 2

Pld exhibits classical Phospholipase D activity regulated by signaling pathways and peaks in expression prior to cellularization. A. HEK-293 cells stably expressing the m3 muscarinic acetylcholine receptor were co-transfected with pCGN:Pld and with ARF1, ARF6 or RhoA. After 24 hours, the cells were stimulated with carbachol, an activator of G-protein coupled muscarinic receptors, and assayed in vivo for PLD activity. The assay is based on the ability of PLD to use primary alcohols, such as butanol, in place of water, in the PC hydrolysis reaction. This leads to the production of phosphatidyl-alcohol (-butanol, PtdBut). Assaying PtdBut is preferable to attempting to quantitate PA, since PA is highly labile and turns over quickly, whereas PtdBut is relatively inert, accumulates over the course of the 30' assay, and thus provides an estimate of the total amount of PLD activity that took place during the assay period. See citation 41 for assay details. The data represent means of triplicate measurements. Standard deviations averaged 3–5%. The experiment is representative of three independent experiments. *, significant difference, p < 0.05. B. Temporal expression of endogenous Pld as analyzed by quantitative PCR. Embryonic stages are given in hours after egg deposition (AED). 0–1 NF, non-fertilized eggs between 0 and 1 hour AED. Cellularization occurs between 2 and 3 h AED during the 14^th mitotic cycle. Pld cDNA concentration is expressed as a ratio of Pld to rp49, a ribosomal RNA expressed at constant levels.