Davis Amanda J, Perera Imara Y, Boss Wendy F
Department of Botany, North Carolina State University, Raleigh, NC 27695, USA.
J Lipid Res. 2004 Sep;45(9):1783-9. doi: 10.1194/jlr.D400005-JLR200. Epub 2004 Jun 21.
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.
肌醇脂激酶已在植物和动物系统中得到广泛研究。然而，重组脂激酶体外研究的主要局限在于纯化蛋白的比活性低和稳定性差。我们的目标是确定环糊精是否能提供有效的底物递送系统并提高脂激酶的比活性。对于这些研究，我们使用了重组拟南芥磷脂酰肌醇磷酸激酶1（At PIPK1）。At PIPK1作为与谷胱甘肽-S-转移酶的融合蛋白产生，并在谷胱甘肽-琼脂糖珠上纯化。使用在α-、β-或γ-环糊精中制备的底物对脂激酶活性进行比较表明，β-环糊精最有效，与在Triton X-100混合胶束中制备的底物相比，可将脂激酶活性提高6倍。我们优化了反应条件，并表明产物可从环糊精处理的重组蛋白中回收，这揭示了一种用于自动化药理筛选测定的潜在方法。
