Partial purification and characterization of phosphatidylinositol kinases from human platelets.
Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCl treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76,000 and 80,000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550,000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.
Kanoh
H
H
Department of Biochemistry, Gifu University School of Medicine, Japan.
Banno
Y
Y
Hirata
M
M
Nozawa
Y
Y
D019870:1-Phosphatidylinositol 4-Kinase; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology* / Q000648:ultrastructure; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol; D010718:Phosphatidylserines / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000097:blood*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011189:Potassium Chloride; D010738:Type C Phospholipases / Q000378:metabolism
D001095:Arachidonic Acids; D010718:Phosphatidylserines; D016718:Arachidonic Acid; D011092:Polyethylene Glycols; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D017830:Octoxynol; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D010738:Type C Phospholipases; D008274:Magnesium