Fig. 7.

Ca²⁺ -dependent phospholipid binding to the native and the mutant PLC δ1. Centrifugation binding assay of PLC δ1 and sucrose loaded PS/PC vesicles in the presence of the indicated concentration of free Ca²⁺ ion. 1μg of the native (A), D653G (B), D706G (C) and D708G (D) mutant protein was incubated with 150 μM PS/PC vesicles (molar ratio = 1:1) in 0.2 ml of 50 mM Hepes, pH 7.0, 100 mM KCl, 2 mM EGTA and various concentrations of CaCl₂ to yield the indicated concentration of free Ca²⁺. The reaction was incubated at 30°C for 15 min. The bound enzyme (pellet fraction) and the free enzyme (supernatant fraction) were separated and quantitated as described under Experimental Procedures.