Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.
PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.
Graziani
A
A
Department of Physiology, Tufts University School of Medicine, Boston, MA 02111.
Ling
L E
LE
Endemann
G
G
Carpenter
C L
CL
Cantley
L C
LC
D019870:1-Phosphatidylinositol 4-Kinase; D000911:Antibodies, Monoclonal; D003600:Cytosol / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase