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 phosphatid ylinositol 3,4,5-trisphosphate (PI(3,4,5)P-3) metabolism, we have discovere d three distinct phosphoinositide-specific phospholipase D (PI-PLD) isozyme s from rat brain, tentatively designated as PI-PLD, PI-PLDb, and PI-PLDc. T hese enzymes convert [H-3]PI(3,4,5)P-3 to generate a novel inositol phospha te, D-myo [H-3]inositol 3,4,5-trisphosphate ([H-3]Ins(3,4,5)P-3) and phosph atidic acid. These isozymes are predominantly associated with the cytosol, a notable difference from phosphatidylcholine PLDs. They are partially puri fied 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)P-3, with a relative potency of PI(3,4,5)P-3 >> p hosphatidylinositol 3-phosphate (PI(3)P) or phosphatidylinositol 4-phosphat e (PI(4)P) > phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-3) > phosphati dylinositol 3,4-bisphosphate (PI(3,4)P-3). In contrast, PI-PLDc preferentia lly utilizes PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P-3, P I(C)P, PI(3,4)P-2, and PI(4,5)P-2. Both PI(3,4)P-2 and PI(4,5)P-2 are poor substrates for all three isozymes, indicating that the regulatory mechanism s underlying these phosphoinositides are different from that of PI(3,4,5)P- 3. None of these enzymes reacts with phosphatidylcholine, phosphatidylserin e, or phosphatidylethanolamine. All three PI-PLDs are Ca2+-dependent. Among them, PI-PLDb and PI-PLDc show maximum activities within a sub-mu M range (0.3 and 0.9 mu M Ca2+ respectively), whereas PI-PLDa exhibits an optimal [ Ca2+] at 20 mu M. In contrast to PC-PLD, Mg2+ has no significant effect on the enzyme activity. All three enzymes require sodium deoxycholate for opti mal activities; other detergents examined including Triton X-100 and Nonide t P-40 are, however, inhibitory. In addition, PI(4,5)P-2 stimulates these i sozymes in a dose-dependent manner. Enhancement in the enzyme activity is n oted only when the molar ratio of PI(4,5)P-2 to PI(3,4,5)P-3 is between 1:1 and 2:1.