Purification and characterization of a phosphatidylinositol kinase from A431 cells.
Purification and characterization of a phosphatidylinositol kinase from A431 cells.
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A431 细胞中磷脂酰肌醇激酶的纯化和表征。
作者：
Duncan H. Walker;Nancy Dougherty;Linda J. Pike
从A431细胞的磷脂酰肌醇激酶已被纯化至接近同质。通过使用层析步骤的组合实现纯化，包括用PI从肝素-琼脂糖柱亲和洗脱酶。由纯化的PI激酶形成的[32 P]PIP的表征表明，该酶在4-位磷酸化肌醇，因此是磷脂酰肌醇4-激酶。该酶具有55，000的亚基重量，如通过SDS凝胶电泳所估计的，并且似乎作为单体具有活性。对酶的流体动力学性质的研究表明，PI激酶结合大量的Triton X-100，并且实际上作为分子量约为120，000的复合物存在于含洗涤剂的溶液中。酶对PI的Km为16 μ M，对ATP的Km为74 μ M。该酶被腺苷抑制，IC 50为100 μ M。这些性质与EGF刺激的A431细胞中的膜结合PI激酶的性质基本相同。因此，数据表明EGF刺激的PI激酶是55，000-Da单体。
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.
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DOI：
10.1073/pnas.84.8.2170
发表时间：
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影响因子：
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DOI：
--
发表时间：
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期刊：
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DOI：
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DOI：
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DOI：
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发表时间：
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期刊：
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影响因子：
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