Identification of multiple phosphoinositide-specific phospholipases D as new regulatory enzymes for phosphatidylinositol 3,4,5-trisphosphate

In the course of delineating the regulatory mechanism underlying phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) metabolism, we have discovered three distinct phosphoinositide-specific phospholipase D (PI-PLD) isozymes from rat brain, tentatively designated as PI-PLDa, PI-PLDb, and PI- PLDc. These enzymes convert [³H]PI(3,4,5)P₃ to generate a novel inositol phosphate, D-myo-[³H]inositol 3,4,5-trisphosphate ([³H]Ins(3,4,5)P₃) and phosphatidic acid. These isozymes are predominantly associated with the cytosol, a notable difference from phosphatidylcholine PLDs. They are partially purified by a three-step procedure consisting of DEAE, heparin, and Sephacryl S-200 chromatography. PI-PLDa and PI-PLDb display a high degree of substrate specificity for PI(3,4,5)P₃, with a relative potency of PI(3,4,5)P₃ >> phosphatidylinositol 3-phosphate (PI(3)P) or phosphatidylinositol 4-phosphate (PI(4)P) > phosphatidylinositol 4,5- bisphosphate (PI(4,5)P₂) > phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂). In contrast, PI-PLDc preferentially utilizes PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P₃, PI(4)P, PI(3,4)P₂, and PI(4,5)P₂. Both PI(3,4)P₂ and PI(4,5)P₂ are poor substrates for all three isozymes, indicating that the regulatory mechanisms underlying these phosphoinositides are different from that of PI(3,4,5)P₃. None of these enzymes reacts with phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine. All three PI-PLDs are Ca²⁺-dependent. Among them, PI-PLDb and PI-PLDc show maximum activities within a sub-μM range (0.3 and 0.9 μM Ca²⁺, respectively), whereas PI-PLDa exhibits an optimal [Ca²⁺] at 20 μM. In contrast to PC-PLD, Mg²⁺ has no significant effect on the enzyme activity. All three enzymes require sodium deoxycholate for optimal activities; other detergents examined including Triton X-100 and Nonidet P- 40 are, however, inhibitory. In addition, PI(4,5)P₂ stimulates these isozymes in a dose-dependent manner. Enhancement in the enzyme activity is noted only when the molar ratio of PI(4,5)P₂ to PI(3,4,5)P₃ is between 1:1 and 2:1.