Purification and Characterization of a Phosphatidylinositol Kinase from A431 Cells

A phosphatidylinositol kinase from A431 cells has been purified to near homogeneity. Purification was achieved through the use of a combination of chromatography steps including affinity elution of the enzyme from a heparin-agarose column with PI. Characterization of the [³²P]PIP formed by the purified PI kinase indicates that the enzyme phosphorylates the inositol on the 4-position and is therefore a phosphatidylinositol 4-kinase. The enzyme has a subunit weight of 55 000 as estimated by SDS gel electrophoresis and appears to be active as a monomer. Studies of the hydrodynamic properties of the enzyme indicate that the PI kinase binds substantial amounts of Triton X-100 and is actually present in detergent-containing solutions as a complex with a molecular weight of approximaely 120000. The K_mof the enzyme for PI is 16 μm and for ATP is 74 μM. The enzyme is inhibited by adenosine with an IC₅₀of 100 μM. These properties are essentially identical with those of the membrane-bound PI kinase in A431 cells which is stimulated by EGF. The data therefore suggest that the EGF-stimulated PI kinase is a 55000-Da monomer.