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 Km 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 Ki of 32 μM. Enzymatic activity was found to be stimulated by Triton X‐100 but inhibited by deoxycholate.
|Number of pages||7|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Oct 1991|