New aspects of formation of 1,2-cyclic phosphates by phospholipase C-δ1 Original Research Article

Phosphoinositide-specific phospholipase C-δ1 (PI-PLC-δ1) cleaves phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2, 1), 5-phosphate (PI-5-P, 2) and 4-phosphate (PI-4-P, 3) to form the mixture of the corresponding 4,5-, 5- and 4-phosphorylated inositol 1,2-cyclic phosphate (IcP) and 1-phosphate (IP) (4–6 and 7–9, respectively). In this work, we have studied the rates of the cleavage and the ratios of the cyclic-to-acyclic phosphate products under various pH and Ca2+ concentration conditions using 31P NMR to monitor the reactions. In agreement with the previous report (Kim et al. Biochim. Biophys. Acta 1989, 163, 177), our results indicate that the IcP/IP ratios strongly depend on the reaction conditions, with the cyclic phosphate products formed predominantly at low pH (pH 5.0) and high calcium concentration (5 mM). Surprisingly, however, we have found that at pH 8.0 and 5 mM Ca2+, PI-5-P rather than PI-4,5-P2 is the most preferred substrate with the highest Vmax. The cleavage of PI-5-P generated also more cyclic phosphate product than the other two substrates. In addition, we have studied the analogous reaction of phosphorothioate analogues of 1 with the sulfur placed in the nonbridging (10) or bridging (13) positions. We have found that the phosphorothioate analogue 10 produced exclusively the cyclic product 11, whereas the analogue 13 afforded exlusively the acyclic product 7. These results are discussed in terms of the mechanism of PI-PLC, where the cyclic product is formed by ‘leaking’ from the active site before its subsequent hydrolysis. The potential significance of the cyclic products in the signaling pathways is also discussed.