Purification and kinetic properties of a soluble phosphatidylinositol-4-phosphate kinase of the bovine adrenal medulla with emphasis on its inhibition by calcium ions.
A phosphatidylinositol-4-phosphate kinase (PIP kinase, EC 2.7.1.68) was purified about 1000-fold from the cytosolic fraction of bovine adrenal medulla by ammonium sulfate precipitation, anion exchange, phosphocellulose and gel permeation chromatography. The PIP kinase preparation was free from other polyphosphoinositide metabolizing activities, and some of its catalytic properties were studied using both membrane-bound and liposomal/micellar phosphatidylinositol 4-phosphate (PIP) as the substrate. Using a native substrate (chromaffin granule ghosts) enriched in PIP, the Km value for MgATP was about 130 microM at 5 mM Mg2+, and optimum activity was obtained at pH 7.2. ADP inhibited the PIP kinase activity in a mixed non-competitive manner. The enzyme was stimulated by Mg2+ and spermidine, whereas free calcium effectively inhibited the PIP kinase activity (I50 at about 0.1 microM Ca2+). This inhibition was independent of calmodulin and CaATP (80 microM) was not a substrate for the PIP kinase. The presence of near intracellular concentrations of potassium decreased the sensitivity to calcium ions (I50 of about 50 microM), but the inhibitory effect was still evident in the physiologically interesting concentration range. The concentration for half-maximal inhibition was three orders of magnitude higher (I50 of about 200 microM) using a liposomal/micellar suspension of purified PIP as the substrate, which demonstrates the importance of using a native substrate in studying the regulatory properties of this enzyme. It is concluded that the high sensitivity of the PIP kinase activity to calcium ions is likely to be physiologically significant, as recently proposed for the phosphatidylinositol (PI) kinase (Husebye, E.S. and Flatmark, T. (1988) Biochim. Biophys. Acta 968, 261-265), and may represent a negative feedback control of the cytosolic calcium concentration. The cationic amphiphile trifluoperazine was a potent inhibitor of PIP kinase activity (I50 at about 15 microM), and may represent a useful tool to study PI kinase selectively in membranes or cells containing both kinases.
Husebye
E S
ES
Department of Biochemistry, University of Bergen, Norway.
Flatmark
T
T
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007202:Indicators and Reagents; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor)
D007202:Indicators and Reagents; D015636:Magnesium Chloride; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D008274:Magnesium; D002118:Calcium