ホスフォリパーゼＣゼータの酵素活性およびカルシウムオシレーション誘発能におけるEFハンドドメインの役割

抄録

Phospholipase C-zeta (PLCζ), a novel isoform of PLC, receives much attention, because it is a strong candidate of the mammalian sperm factor that is driven into the egg cytoplasm upon sperm-egg fusion at fertilization and induces Ca²⁺ oscillations leading to egg activation. We have synthesized recombinant PLCζ using baculovirus/Sf9-cell expression system and showed that PLCζ can induce Ca²⁺ oscillations at low concentrations probably via IP₃-induced Ca²⁺ release when injected into mouse eggs. We also showed that PLCζ has such a high Ca²⁺-sensitivity of PLC activity that the enzyme can be active in resting cells at ∼100 nM Ca²⁺. The structure-function relationship was examined by mutational analysis. Deletion of EF1 and EF2 of N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate-hydrolysing activity in vitro and loss of Ca²⁺ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 little affected the Ca²⁺-sensitivity of PLC activity, while deletion of EF1 to EF3 caused 12-fold elevation of EC50 of Ca²⁺ concentration. Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca²⁺ sensitivity. Deletion of four EF-hand domains or C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisite for the PLCζ activity. [Jpn J Physiol 55 Suppl:S73 (2005)]