Fig. 9.

Binding of Ca²⁺ and PS to the D653, 706, 708G mutant PLC δ1. Binding of 0.15 μM the D653/D706/D708G mutant enzyme with indicated concentration of free ⁴⁵Ca²⁺ (total 1.5 × 107 cpm) in 0.1 ml buffer containing 50 mM Hepes, pH 7.0, 100 mM KCl, and 10 μM EGTA and 150 μM PS/PC vesicles containing 0 (diamond) and 35 (circle) mol% of PS, the determination of bound Ca²⁺ were carried as described in Experimental Procedures (A). Centrifugation binding assay of the D653/D706/D708G mutant enzyme and sucrose loaded PS/PC vesicles in the presence of the indicated concentration of free Ca²⁺ ion. Binding of 1μg protein to150 μM PS/PC vesicles (molar ratio = 1:1) in the presence of indicated concentration of free Ca²⁺ was carried as described (Fig. 6 and Experimental Procedures) (B).