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 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.
Walker
D H
DH
Department of Biological Chemistry, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110.
Dougherty
N
N
Pike
L J
LJ
D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine / Q000494:pharmacology; D002413:Cations, Divalent; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000494:pharmacology; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology
D002413:Cations, Divalent; D010716:Phosphatidylinositols; D012265:Ribonucleotides; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D000241:Adenosine