Characterization of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in human neutrophils.
Phosphodiesteric cleavage of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) is required for transmembrane signaling by chemoattractants in human polymorphonuclear leukocytes (PMN). Considering the importance of PtdIns-4,5-P2 as a reservoir for second messenger substances, we have characterized the enzyme system that synthesizes this phospholipid in human PMN, consisting of kinases for phosphatidylinositol (PtdIns) and phosphatidylinositol-4-phosphate (PtdIns-4-P). The preferred phosphate donor for both enzymes was ATP as compared with GTP. The respective Km for ATP for PtdIns kinase and PtdIns-P kinase were 0.049 +/- 0.013 and 0.062 +/- 0.005 mM and for GTP were 0.242 +/- 0.016 and 0.186 +/- 0.037 mM. PtdIns stimulated the activity of PtdIns kinase to a greater extent than PtdIns-4-P kinase. PtdIns-4-P inhibited the activity of detergent-solubilized PtdIns kinase and stimulated particulate PtdIns-4-P kinase, whereas both enzymes exhibited substrate inhibition to PtdIns-4,5-P2. Mg2+ was the preferred cation for both enzymes, but the apparent Km values (4.1 +/- 0.9 mM for PtdIns kinase and 1.0 +/- 0.7 mM for PtdIns-4-P kinase) were significantly different (p less than 0.005). Mn2+ partially substituted for Mg2+, and both enzymes were inhibited by Ca2+. The polyamine spermine stimulated PtdIns-4-P kinase activity to a greater extent and at lower concentrations than PtdIns kinase. PtdIns kinase was easily solubilized in both Triton X-100 and Nonidet P-40, whereas PtdIns-4-P kinase remained in a detergent-nonextractable membrane fraction. These findings demonstrate that the enzyme system in human PMN that forms PtdIns-4,5-P2 is composed of two distinct enzymes with similar characteristics.
Pike
M C
MC
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109.
Arndt
C
C
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013096:Spermine / Q000494:pharmacology
D003902:Detergents; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C037178:phosphatidylinositol 4-phosphate; D013096:Spermine; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D008274:Magnesium; D002118:Calcium