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J. Lipid Res.
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A more recent version of this article appeared on September 1, 2004

Papers In Press, published online ahead of print June 21, 2004
J. Lipid Res., doi:10.1194/jlr.D400005-JLR200
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Submitted on April 23, 2004
Revised on June 10, 2004
Accepted on June 11, 2004

Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity

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

Department of Botany, North Carolina State University, Raleigh, NC 27695-7612

Corresponding Author: 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 phosphatidylinositol phosphate kinase 1 (AtPIPK1). AtPIPK1 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 six-fold compared to 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.


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This article has been cited by other articles:


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A. J. Davis, Y. J. Im, J. S. Dubin, K. B. Tomer, and W. F. Boss
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Y. J. Im, A. J. Davis, I. Y. Perera, E. Johannes, N. S. Allen, and W. F. Boss
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