Enzymatic synthesis of pyrene-labeled polyphosphoinositides and their behavior in organic solvents and phosphatidylcholine bilayers

A method is reported for the synthesis of pyrene-labeled analogues of phosphatidylinositol 4-phosphate (Pyr-PIP) and phosphatidylinositol 4,5-bisphosphate (Pyr-PIP,) from sn-2-(pyrenyl-decanoy1)phosphatidylinositol (Pyr-PI) using partially purified PI and PIP kinase preparations. Phos-phorylation of Pyr-PI and Pyr-PIP was extensive (more than 50%) provided that the ATP concentration was high and that stabilizing agents such as sucrose and polyethylene glycol were present in the incubation medium. Pyr-PIP and Pyr-PIP, were isolated by chromatography on immobilized neomycin. The identity of the products was established by thin-layer chromatography, UV-absorption spectroscopy, and spectro-fluorometry. The pyrene excimer/monomer fluorescence technique revealed that, in contrast to Pyr-PI, Pyr-PIP and Pyr-PIP, formed clusters in organic solvents. By use of the same technique for model membranes, it was shown that in phosphatidylcholine bilayers the collision frequency of the three fluorescent phos-phoinositides decreased in the order PI > PIP > PIP2. Addition of Ca2+ a t concentrations above 0.1 mM increased the collision frequency of Pyr-PIP2 and, to a much lesser extent, Pyr-PIP; Ca2+ had no effect on Pyr-PI.