Manganese-stimulated phosphatidylinositol 4-kinase from Dunaliella parva: purification and characterization

Abstract

A soluble phosphatidylinositol (PI) kinase (ATP:phosphatidylinositol phosphotransferase, EC 2.7.1.67) was partially purified from Dunaliella parva. Analyses of the enzyme product and its water-soluble moiety, obtained by deacylation and removal of the glycerol, revealed that the enzyme is a PI 4-kinase. An apparent molecular weight of about 500 000 was determined by size exclusion chromatography and glycerol density gradient centrifugation. Chromatofocusing and isoelectric focusing displayed p I values of 5.5 and 6.6, respectively. The kinase required divalent cations such as Mg 2+ (optimum at 30 mM) and Ca 2+ (optimum at 10 mM); however, in the presence of Mn 2+ ions (optimum at 10 mM) the activity was about 2.5-fold higher. Furthermore, Mn 2+ and Mg 2+ (or Ca 2+) ions activated synergistically; at a constant Mg 2+ concentration (2 mM), Mn 2+ ions stimulated the activity up to about 20-fold, whereas at a constant Mn 2+ concentration (10 mM) Mg 2+, ions stimulated up to about 4-fold. The temperature optimum of the enzyme activity increased from 28°C at 30 mM MgCl 2 to 42°C at 2 mM MgCl 2 and 10 mM MnCl 2, whereas the pH optimum of 7.8 did not change. The apparent K m value for ATP depended on the divalent cation; at 2 mM MgCl 2 and 10 mM MnCl 2, a K m value of 390 μM was determined, whereas at 30 mM MgCl 2 the value was 1.7 mM. The activity of the lipid kinase depended on the presence of a surfactant; optimum concentration of Triton X-100 was 3.7 mM (235 μM PI, 2 mM MgCl 2 and 10 mM MnCl 2). The apparent K m value for PI was determined as 55 and 27 μM at 1.5 and 0.75 mM Triton X-100, respectively, which corresponds to a surface concentration of PI in the mixed micelles of about 3.6 mol%. Several nucleoside triphosphates as well as ADP, AMP, adenosine and phosphatidylethanolamine were shown to inhibit the enzyme competitively.