Renaturation of Phosphatidylinositol 4- and Phosphatidylinositol 4-Phosphate 5′-Kinases Following Polyacrylamide Gelelectrophoresis in Presence of SDS. Studies on their Substrate Binding Requirements Using Synthetic Substrate Analogues

Abstract

Phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4 phosphate (PtdIns4P) 5′-kinase catalyze the phosphorylation of PtdIns in two subsequent steps leading to the formation of PtdIns(4,5)P₂. From bovine brain, two membrane bound PtdIns 4-kinases were separated by hydroxyapatite chromatography and identified as type 2 and type 3 isoforms (see Vereb et al., p. 167 in this volume). In parallel, also the PtdIns4P 5′-kinase was eluted from the same column with three activity maxima. A new renaturation procedure has been developed which allows the determination of the apparent molecular weight of both lipid kinases following polyacrylamide gelelectrophoresis in presence of SDS (SDS-PAGE). Furthermore, we explored substrate binding requirements using synthetic substrate analogues with the aim to determine structural features on PtdIns and PtdIns4P which are essential for the function as substrate for the respective kinases.