| Research Abstract |
Phosphatidylinositol 3-kinase (Pl 3-kinase) is a key enzyme in the signaling pathways of 3-phosphorylated polyphosphoinositides. The major substrate of Pl 3-kinase, in vivo, is assumed to be phosphatidylinositol 4, 5-bisphosphate (Pl 4, 5-P_2), generating Pl 3, 4, 5-P_3, a putative second messenger to play important roles in signal transduction. In vitro, the enzyme phosphorylates Pl, Pl 4-P and Pl 4, 5-P_2, The natural Pl purified from animal tissues, which is an excellent substrate of Pl 3-kinase, contains arachidonate at the sn-2 position. On the other hand, we found that synthetic Pls with saturated fatty acid of carbon number 18 and 20 at the sn-2 position, were not phosphorylated by the enzyme. In order to evaluate the phosphorylation reaction with Pl 3-kinase, we optimized the sn-2 fatty acids of Pl.
A mixture of Pls (Pl_<C4>, Pl_<C6>, Pl_<C8>, Pl_<C10>, Pl_<C12>, Pl_<C14>, Pl_<C16>) with various saturated fatty acids at the sn-2 center was synthesized from an equimolar mixture of seven carboxylic acids (C4, C6, C8, C10, C12, C14, C16), and was subjected to the enzymatic reaction with Pl 3-kinase. The product was analyzed by negative ion fast atom bombardment (FAB) mass spectrometry using a matrix (triethanolamine : glycerol = 3 : 1). The spectrum showed the [M-H] ion peaks of monophosphorylated Pl_<C4>, Pl_<C6>, Pl_<C8> and Pl_<C10> analogs. The phosphorylated ions derived from the longer chain analogs such as Pl_<C16> were detected only at trace levels. Short sn-2 fatty acid analogs such as Pl_<C4>, Pl_<C8> were also shown to be excellent substrates of Pl 3-kinase by independent phosphorylation of synthetic Pls (Pl_<C2>, Pl_<C4>, Pl_<C8>, Pl_<C12>, Pl_<C16>, Pl_<C18>) with Rl 3-kinase and [gamma-^<32>P]-ATP.
These results are applicable for the design of artificial second messenger molecules, enzyme inhibitors, affinity probes to find specific binding proteins and photoaffinity labeling probes.
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