Phosphatidylinositol 4-kinases in plasma membranes and Affinity partitioning of liposomes: a model system for membrane purification

Sammanfattning: Polyphosphoinositides, the different phosphorylated variants of the phospholipid phosphatidylinositol (PtdIns), are used in a wide array of intracellular mechanisms in eukaryotic cells. The phosphorylation of PtdIns at carbon D4, catalyzed by phosphatidylinositol 4-kinase, is an important step in the synthesis of several of these molecules. The presence of different isoenzymes of PtdIns 4-kinase in rat liver plasma membranes and their further distribution in plasma membrane domains was examined. The wortmannin-sensitive kinase PI4K230 was located in blood sinusoidal plasma membrane domains (ca 25% of the total cell content), while PI4K92, also wortmannin-sensitive, could not be detected in plasma membranes. Together with earlier observations of the wortmannin sensitivity of the PtdIns 4,5-P2 synthesis during agonist stimulation, this indicates that PI4K230 is involved in transmembrane signaling. The presence of wortmannin-sensitive PtdIns 4-kinase activity in plasma membranes was confirmed by experiments on rabbit lacrimal glands. A third isoenzyme, PI4K55, was mainly found in exocytic vesicles, but a small part (less than 10%) was located in lateral plasma membrane domains. Such a distribution might suggest a role in intracellular membrane transport, but possibly also in the maintenance of cell-cell adhesion complexes. PtdIns 4-kinase activity was examined in spinach plasma membranes as well. Two different fractions containing PtdIns 4-kinase activity were resolved by chromatography. The molecular weights of the corresponding isoenzymes were determined to be 120 and 65 kDa respectively, and they were shown to differ in their sensitivity to wortmannin and the response to divalent cations. Both isoenzymes reacted with an antibody directed against mammalian a isoforms thereby implicating a relation to the mammalian PI4K230. Affinity partitioning in poly(ethylene glycol)/dextran two-phase systems using wheat germ agglutinin coupled to dextran as affinity ligand is an efficient and highly specific method to purify plasma membranes from different sources, as shown, for instance, in the work concerning PtdIns 4-kinase. In order to extend the method to be of general use in the purification of membranes, it was necessary to explore basic requirements for the affinity partitioning of subcellular membranes. To this end, biotinylated liposomes were used as model membranes. Such liposomes could be specifically redistributed to the dextran-rich bottom phase by avidin-dextran, but only if millimolar concentrations of Li2SO4 was included in the system. As little as 1 or 2 biotin residues per liposome was sufficient to cause this redistribution. Furthermore, negative surface charges affected the redistribution by interfering with the biotin-avidin binding, but not with the partitioning of the liposome/dextran conjugate per se. Thus, basic knowledge was gained setting the stage for future development of affinity partitioning as a tool for the purification of biological membranes.