Figure 3.
U-73122 reduces the activity of a recombinant plant PI-PLC, whereas the inactive analogue U-73343 has no effect on the activity. The PI-PLC clone isolated from a guard-cell-enriched cDNA library of N. rustica was expressed as a recombinant GST fusion protein. This protein appeared as a discrete band when analyzed by SDS/PAGE [Inset, lane 1, molecular mass markers of 200, 116, 97, 66, and 45 kDa; lane 2, 1 μg of affinity-chromatography-purified GST-PI-PLC (arrowhead)]. After a 15-min preincubation with increasing concentrations of U-73122 (closed circles) or U-73343 (open circles), the activity of the recombinant plant PI-PLC was determined with phosphatidylinositol 4,5-bisphosphate as the substrate. Values were expressed as a percentage of the activity in the absence of U-73122 or U-73343; 100% of PI-PLC activity represents 300–500 nmol⋅min−1 per mg of protein.