MANGANESE-STIMULATED PHOSPHATIDYLINOSITOL 4-KINASE FROM DUNALIELLA-PARVA - PURIFICATION AND CHARACTERIZATION

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 pi values of 5.5 and 6.6, respectively. The kinase required divalent cations such as Mg2+ (optimum at 30 mM) and Ca2+ (optimum at 10 mM); however, in the presence of Mn2+ ions (optimum at 10 mM) the activity was about 2.5-fold higher. Furthermore, Mn2+ and Mg2+ (or Ca2+) ions activated synergistically; at a constant Mg2+ concentration (2 mM), Mn2+ ions stimulated the activity up to about 20-fold, whereas at a constant Mn2+ concentration (10 mM) Mg2+, ions stimulated up to about 4-fold. The temperature optimum of the enzyme activity increased from 28 degrees C at 30 mM MgCl2 to 42 degrees C at 2 mM MgCl2, and 10 mM MnC1(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 MgCl2, and 10 mM MnCl2, a K-m value of 390 mu M was determined, whereas at 30 mM MgCl2 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 mu M PI, 2 mM MgCl2 and 10 mM MnCl2). The apparent K-m value for PI was determined as 55 and 27 mu M 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.