STRUCTURAL FEATURES OF GPI-SPECIFIC PHOSPHOLIPASE-D REVEALED BY PROTEOLYTIC FRAGMENTATION AND CA2-STUDIES( BINDING)

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abu ndant in plasma and is potentially capable of degrading the anchor uti lized by many cell surface proteins. The goal of this work was to stud y structural features of the GPI-PLD that might be involved in regulat ion of its activity. Trypsin cleaved the 100-110 kDa GPI-PLD polypepti de into three major fragments (two of similar to 40 kDa and a carboxyl -terminal fragment of 30 kDa) which were relatively resistant to furth er proteolysis. Pretreatment of the GPI-PLD with chelators resulted in complete degradation. During the cleavage process the GPI-PLD enzymat ic activity increased similar to 3-4-fold but no other major change in its properties (e.g. inhibition by chelators and lipids, thermal stab ility, oligomerization, etc.) was observed. Intact or trypsinized GPI- PLD bound Ca-45(2+) (similar to 5.5 ions/molecule GPI-PLD); K-d simila r to 16.1 mu M as determined by equilibrium dialysis) which could not be blocked by the addition of other divalent metal ions. However, inhi bition of enzymatic activity by divalent cation chelators appeared to involve removal of bound Zn2+ rather than Ca2+. A metal analysis of GP I-PLD revealed similar to 5 and 10 atom/molecule of calcium and zinc, respectively. The data suggest that the predicted integrin E-F hand-li ke sites in GPI-PLD are functional but not directly involved in enzyma tic activity.