Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells

Abstract

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-kinase) in carrot (Daucus carofa L.) suspension culture cells (C.M. Okpodu, W. Cross, W.F. Boss 119901 Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-1 O0 gel filtration column chromatography. The M, of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. l h e isolated lipid kinase phosphorylated PI but not lysophosphatidylinosito1 or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-lOO/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mgz+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mgz+. Calcium from 0.01 p~ to 1 mM had no effect on the enzyme activity. The K,,, of the enzyme for ATP was estimated to be between 400 and 463 p~. The enzyme was inhibited by adenosine (100 p~) ; however, ADP (up to 100 p~) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast.

Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms

“…In plant cells, PtdIns kinase activity was originally found to be associated with plasma membranes (Sandelius and Sommarin, 1986;Sommarin and Sandelius, 1988;Kamada and Muto, 1991), and has subsequently been found in the cytoskeleton (Tan and Boss, 1992;Xu et al, 1992), nuclei (Hendrix et al, 1989), and soluble fraction (Steinert et al, 1994;Okpodu et al, 1995). Plant PtdIns 4-kinases have been partially purified from the soluble fraction of cells of the unicellular alga Dunaliella parva (Steinert et al, 1994) and carrot (Okpodu et al, 1995), and from Catharanthus roseus plasma membranes (Hanenberg et al, 1995).…”

“…Plant PtdIns 4-kinases have been partially purified from the soluble fraction of cells of the unicellular alga Dunaliella parva (Steinert et al, 1994) and carrot (Okpodu et al, 1995), and from Catharanthus roseus plasma membranes (Hanenberg et al, 1995). The soluble carrot enzyme has an apparent molecular mass of approximately 80 kD, both on gel filtration and after renaturation following SDS-PAGE (Okpodu et al, 1995), whereas both the D. parva enzyme and the solubilized C. roseus plasma membrane enzyme eluted at 500 kD or above on gel filtration (Steinert et al, 1994;Hanenberg et al, 1995), indicating that these enzymes form large complexes.…”

“…There is a paucity of kinetic data on plant PtdIns kinases, but the C. roseus enzyme shows low K m values for ATP and PtdIns similar to animal type II kinases (Hanenberg et al, 1995), whereas the carrot enzyme exhibits a K m for ATP in the type III range (Okpodu et al, 1995). Both of the isolated soluble enzymes are inhibited by adenosine and require comparatively high concentrations of Mg 2ϩ (30-40 mm) for maximum activity (Steinert et al, 1994;Okpodu et al, 1995).…”

Phosphatidylinositol 4-Phosphate Negatively Regulates Chloroplast Division in Arabidopsis

“…In addition to being important for signaling as a direct substrate for phospholipase C (Gonorazky et al, 2010) or as a precursor for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] (Berridge and Irvine, 1989), PI4P itself acts as a signaling lipid (Jung et al, 2002;Preuss et al, 2006;Chapman and Goring, 2011). PI4P is synthesized by phosphatidylinositol 4-kinase (PI4K), and PI4K activities are present in many cellular compartments, including the plasma membrane (Sommarin and Sandelius, 1988), cytosol (Okpodu et al, 1995), cytoskeleton (Xu et al, 1992), and nucleus (Hendrix et al, 1989). The existence of PI kinase activity was reported in chloroplast envelope membranes, and phosphatidylinositol 3-phosphate (PI3P) and PI4P were identified as its products (Bovet et al, 2001).…”

Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro

“…In plant cells, PI 4-kinase activity has been detected in different tissues including plasma membranes [27], cytosol [28], cytoskeleton [16] and nuclei [29]. The PI 4-kinase activity present in the plasma membrane of carrot cells responded to osmotic stress, cell wall-digesting enzymes, and light [30].…”