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Journal of Lipid Research, Vol. 45, 1783-1789, September 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity

Amanda J. Davis, Imara Y. Perera and Wendy F. Boss¹

Department of Botany, North Carolina State University, Raleigh, NC 27695

¹ To whom correspondence should be addressed. e-mail: wendy_boss{at}ncsu.edu

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 -, ß-, or -cyclodextrin indicated that ß-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.

Abbreviations: AtPIPK1, Arabidopsis thaliana phosphatidylinositol phosphate kinase 1; CD, ßCD, and CD, -, ß-, and -cyclodextrin; GST, glutathione-S-transferase; NBD-PtdInsP, D(+)-sn-1-O-[1-[6'-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl]amino]-hexanoyl]-2-O-hexadecanoylglyceryl D-myo-phosphatidylinositol phosphate; PtdInsP, phosphatidylinositol phosphate; PtdIns(4,5)P₂, phosphatidylinositol-(4,5)-bisphosphate

Supplementary key words Arabidopsis thaliana • automated assay • inositol lipids

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