Purification and characterization of phosphoinositide 3-kinase from rat liver.
Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.
Carpenter
C L
CL
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Duckworth
B C
BC
Auger
K R
KR
Cohen
B
B
Schaffhausen
B S
BS
Cantley
L C
LC
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D019869:Phosphatidylinositol 3-Kinases; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor; D013379:Substrate Specificity
D000957:Antigens, Viral, Tumor; D003902:Detergents; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010743:Phospholipids; D011956:Receptors, Cell Surface; D008345:Manganese; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D008274:Magnesium