Purification and chemical modification of a phosphatidylinositol kinase from sheep brain

A membrane‐bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500‐fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X‐100, ammonium sulphate fractionation and a number of ion‐exchange and gel‐filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol · min⁻¹· mg⁻¹. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 ± 10 kDa by HPLC gel filtration in the presence of Triton X‐100. Kinetic measurements have shown that the apparent K_m value of PtdIns kinase for the utilisation of PtdIns is 22 μM and for ATP 67 μM. Mg²⁺ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg²⁺. In addition to adenosine and ADP, the 2′(3′)‐O‐(2,4,6‐trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a K_i of 32 μM. Enzymatic activity was found to be stimulated by Triton X‐100 but inhibited by deoxycholate.