title: PI3 kinase enzymology on fluid lipid bilayers.
abstract: We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Debjit
authors.initials: D
authors.lastname: Dutta
authors.forename: Abigail
authors.initials: A
authors.lastname: Pulsipher
authors.forename: Wei
authors.initials: W
authors.lastname: Luo
authors.forename: Muhammad N
authors.initials: MN
authors.lastname: Yousaf
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D057075:Enzyme Assays; D036681:Fluorescence Recovery After Photobleaching; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases