Characterization and purification of membrane-associated phosphatidylinositol-4-phosphate kinase from human red blood cells.
The membrane-bound form of phosphatidylinositol-4-phosphate (PtdInsP) kinase was purified 4,300-fold from human red blood cells to a specific activity of 117 nmol min-1 mg-1. Although this enzyme copurified with red blood cell membranes, it was solubilized by high salt extraction in the absence of detergent indicating that it is a peripheral membrane protein. The major protein seen in the most purified preparation migrated at 53,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major PtdInsP kinase activity in this preparation was also coincident with this 53,000-dalton band upon renaturation of activity from SDS-PAGE. To test further whether the 53,000-dalton protein contained PtdInsP kinase activity, antibodies were prepared against the gel-purified 53,000-dalton protein. This antiserum was able to precipitate both the 53,000-dalton peptide and PtdInsP kinase activity from red blood cell membranes. The apparent size of the native enzyme in the most purified preparation was determined to be 150,000 +/- 25,000 daltons by gel filtration. This PtdInsP kinase activity was at least 100-fold more active in phosphorylating PtdInsP than phosphatidylinositol and was easily separated from the red cell membrane phosphatidylinositol kinase by salt extraction. Analysis of the reaction product, phosphatidylinositol 4,5-bisphosphate, indicates that the enzyme phosphorylates phosphatidylinositol 4-phosphate specifically at the 5'-hydroxyl of the inositol ring. The apparent Km for ATP was 2 microM, and the concentrations of Mg2+ and Mn2+ giving half-maximal activity were 2 and 0.2 mM, respectively. Mg2+ supported 3-fold higher activity than Mn2+ at optimal concentrations. The enzymatic activity was inhibited by its product, phosphatidylinositol 4,5-bisphosphate and enhanced by phosphatidylserine.
Ling
L E
LE
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Schulz
J T
JT
Cantley
L C
LC
D000255:Adenosine Triphosphate / Q000502:physiology; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology* / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008565:Membrane Proteins / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000502:physiology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D011233:Precipitin Tests; D012967:Sodium Dodecyl Sulfate
D002413:Cations, Divalent; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase