Phosphoinositides interact with the polyanion-binding site. (A) Mutation of the polyanion-binding site, but not the distal site, reduces binding to PtdIns(4,5)P2. PtdIns(4,5)P2 was spotted at the indicated amounts on a hydrophobic membrane along with a phosphatidylcholine control. Membrane strips were incubated with IDEwt, the polyanion site mutant IDEK898A,K899A,S901A, or the distal site mutant IDEY609F at the amounts indicated. Bound enzyme was visualized by incubation with anti-IDE antibody. (B) Coomassie blue-stained gel indicating the same amount of the three IDE constructs used for membrane binding. Amounts of IDE for the trials were all within 15.5% of the mean. (C) Displacement of TNP-ATP from the IDE polyanion-binding site by PtdIns(4,5)P2. IDE (66 µg) was mixed with 10 µM TNP-ATP in 50 mM Tris buffer, pH 7.4, and the fluorescent spectra were recorded on an LS 55 Luminescence Spectrometer (PerkinElmer) over the indicated wavelengths. Also shown are the fluorescent spectra of IDE alone, TNP-ATP alone, and IDE with TNP-ATP in the presence of the indicated concentration of PtdIns(4,5)P2.