Assaying phosphoinositide phosphatases.
The roles of phosphoinositide second messengers as signaling molecules in a vast array of cellular processes including cell growth, metabolism, vesicular transport, programmed cell death, and responses to extracellular signals are only beginning to be understood. The recent identification of novel phosphoinositide signaling molecules underscores the need for methodology with which to characterize the enzymes responsible for regulating cellular phosphoinositide levels. One of the ways in which cells control these lipids is through dephosphorylation by phosphoinositide phosphatases, which oppose and regulate the actions of phosphoinositide kinases. We describe herein two rapid and simple assays for characterizing phosphoinositide phosphatases that can be used to provide a basis for understanding the activity and specificity of these enzymes.
Taylor
Gregory S
GS
Department of Pharmacology, University of California San Diego, La Jolla, USA.
Dixon
Jack E
JE
D004396:Coloring Agents; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D010710:Phosphates / Q000032:analysis; D010716:Phosphatidylinositols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012394:Rosaniline Dyes / Q000032:analysis / Q000737:chemistry; D004718:Saccharomycetales / Q000201:enzymology; D015290:Second Messenger Systems; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D004396:Coloring Agents; D005456:Fluorescent Dyes; D010710:Phosphates; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; C005095:malachite green; D010744:Phosphoric Monoester Hydrolases; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C099588:myotubularin