title: Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity.
abstract: Inositol lipid kinases have been studied extensively in both plant and animal systems. However, major limitations for in vitro studies of recombinant lipid kinases are the low specific activity and instability of the purified proteins. Our goal was to determine if cyclodextrins would provide an effective substrate delivery system and enhance the specific activity of lipid kinases. For these studies, we have used recombinant Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (At PIPK1). At PIPK1 was produced as a fusion protein with glutathione-S-transferase and purified on glutathione-Sepharose beads. A comparison of lipid kinase activity using substrate prepared in alpha-, beta-, or gamma-cyclodextrin indicated that beta-cyclodextrin was most effective and enhanced lipid kinase activity 6-fold compared with substrate prepared in Triton X-100-mixed micelles. We have optimized reaction conditions and shown that product can be recovered from the cyclodextrin-treated recombinant protein, which reveals a potential method for automating the assay for pharmacological screening.
authors.affiliation: Department of Botany, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Davis
authors.forename: Imara Y
authors.initials: IY
authors.lastname: Perera
authors.forename: Wendy F
authors.initials: WF
authors.lastname: Boss
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D003505:Cyclodextrins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D003505:Cyclodextrins; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase