Walker D H, Dougherty N, Pike L J
Department of Biological Chemistry, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1988 Aug 23;27(17):6504-11. doi: 10.1021/bi00417a046.
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 approximately 120,000. The Km of the enzyme for PI is 16 microM and for ATP is 74 microM. The enzyme is inhibited by adenosine with an IC50 of 100 microM. 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 55,000-Da monomer.
来自A431细胞的一种磷脂酰肌醇激酶已被纯化至接近均一。纯化是通过一系列色谱步骤实现的，包括用磷脂酰肌醇（PI）从肝素 - 琼脂糖柱上亲和洗脱该酶。对纯化的PI激酶形成的[32P]磷脂酰肌醇磷酸（PIP）的表征表明，该酶使肌醇的4位磷酸化，因此是一种磷脂酰肌醇4 - 激酶。通过SDS凝胶电泳估计，该酶的亚基分子量为55,000，并且似乎以单体形式具有活性。对该酶流体动力学性质的研究表明，PI激酶结合大量的曲拉通X - 100，并且实际上在含去污剂的溶液中以分子量约为120,000的复合物形式存在。该酶对PI的Km为16微摩尔，对ATP的Km为74微摩尔。该酶被腺苷抑制，IC50为100微摩尔。这些性质与A431细胞中受表皮生长因子（EGF）刺激的膜结合PI激酶的性质基本相同。因此，数据表明受EGF刺激的PI激酶是一种55,000道尔顿的单体。
