Figure 1. Identification of D. discoideum PLB enzyme activity.

(A) Dictyostelium lysate (10 μg) was incubated in the presence of GTPγS (30 μM) and myrARF1 (10 μM) at 37 °C for the indicated times. Water-soluble metabolites were separated into choline and phosphorylated metabolites. The results are expressed as d.p.m. of products liberated. Error bars represent range of two points (smaller than symbol in most instances). (B) Dictyostelium lysate was sedimented at 100000 g to separate membranes from cytosol. Lysate (40 μg), membranes (16 μg), and cytosol (18 μg) were assayed using didecanoyl-PC substrate (material used as cell equivalent). Incubations were performed for 60 min at 37 °C in the presence and absence of GTPγS and myrARF1. Water-soluble metabolites were separated into choline and phosphorylated metabolites. The results are expressed as the percentage of PC hydrolysed. Error bars represent range of two points. (C) Dictyostelium lysate (DD; 80 μg), and PLC from B. cereus (BC; 1 units/ml) were assayed using [¹⁴C]didecanoyl-PC substrate (labelled with ¹⁴C in both fatty acid chains). Counts were added to give 92000 d.p.m. per reaction. Incubations were performed for 60 min at 37 °C in the absence of any stimulus. The organic phase was dried, re-solubilized in chloroform, and separated using TLC. Radioactivity on the TLC plate was determined using a phosphorimager. DAG (diacylglycerol), FFA (NEFA) and LPC are indicated. con, substrate alone.