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

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Abstract
A method is reported for the synthesis of pyrene-labeled analogues of phosphatidylinositol 4-phosphate (Pyr-PIP) and phosphatidyalinositol 4,5-bisphosphate (Pyr-PIP2) from sn-2-(pyrenyl-decanoyl)phosphatidylinositol (Pyr-PI) using partially purified PI and PIP kinase preparations. Phosphorylation of Pyr-PI and Pyr-PIP was extensive (more than 50%) provided that the ATP concentration was high and that stabilizing agent such as sucrose and polyethylene glycol were present in the incubation medium. Pyr-PIP and Pyr-PIP2 were isolated by chromatography on immobilized neomycin. The identity of the products was established by thin-layer chromatography, UV-adsorption spectroscopy, and spectrofluorometry. The pyrene excimer/monomer fluorescence technique revealed that, in contrast to Pyr-PI, Pyr-PIP and Pyr-PIP2 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 phosphoinositides decreased in the order PI > PIP > PIP2. Addition of Ca2+ at 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.