Catalytic properties of a purified phosphatidylinositol-4-phosphate kinase from rat brain.
A phosphatidylinositol-4-phosphate (PIP) kinase activity was purified from rat brain extract through several chromatographic steps to yield an active preparation (specific activity 1 mumol of 32P incorporated into phosphatidylinositol 4,5-bisphosphate/min per mg of protein) with an apparent molecular size of 100-110 kDa in the native form. The isolated PIP kinase required Mg2+ (optimally 20-30 mM) for its activity and was not influenced by Ca2+. The enzyme used ATP (Km 25 microM) and GTP (Km 133 microM) as phosphate sources and appeared specific for PIP (Km 3.3 micrograms/ml) as the lipid substrate. The PIP-phosphorylation reaction was inhibited by micromolar concentrations of heparin [ID50 (concn. giving 50% inhibition) 2 micrograms/ml] and the flavonoid quercetin (ID50 0.2 microM). Whereas heparin behaves as a competitive inhibitor to PIP, quercetin was competitive towards ATP (or GTP). Phosphorylation of the preparation by a highly active purified protein kinase C did not detectably alter PIP kinase activity. Whereas 12-O-tetradecanoylphorbol acetate and various phospholipids had no effect, phosphatidylserine elicited a dose-dependent activation of PIP activity. This suggests that a phosphatidylserine-PIP kinase interaction may be considered as a possible regulatory process at the cell-membrane level.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity
D009711:Nucleotides; D010743:Phospholipids; D013096:Spermine; D006493:Heparin; D011794:Quercetin; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D008274:Magnesium