title: Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis.
abstract: The activity of phospholipase A(2) (PLA(2)) which catalyzes the hydrolysis of phospholipids into free fatty acids and lysolipids, depends on the structure and thermodynamic state of the membrane. To further understand how the substrate conformation correlates with enzyme activity, model systems that are based on time-resolved membrane microscopy are needed. We demonstrate a methodology for preparing and investigating the dynamics of fluid supported phospholipid membranes hydrolyzed by snake venom PLA(2). The method uses quantitative analysis of time-lapse fluorescence images recording the evolution of fluid bilayer islands during hydrolysis. In order to minimize interactions with the support surface, we use double bilayer islands situated on top of a complete primary supported membrane prepared by hydration of spincoated lipid films. Our minimal kinetic analysis describes adsorption of enzyme to the membrane in terms of the Langmuir isotherm as well as enzyme kinetics. We use two related models assuming hydrolysis to occur either at the perimeter or at the surface of the membrane island. We find that the adsorption constant is similar for the two cases, while the estimated turnover rate is markedly different. The PLA(2) concentration series is measured in the absence and presence of beta-cyclodextrin which forms water soluble complexes with the reaction products. The results demonstrate the versatility of double bilayer islands as a membrane model system and introduces a new method for quantifying the kinetics of lipase activity on membranes by directly monitoring the evolution in substrate morphology.
authors.affiliation: MEMPHYS, Center for Biomembrane Physics, Physics Department, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. adam@memphys.sdu.dk
authors.forename: Adam Cohen
authors.initials: AC
authors.lastname: Simonsen
authors.forename: Uffe Bernchou
authors.initials: UB
authors.lastname: Jensen
authors.forename: Per Lyngs
authors.initials: PL
authors.lastname: Hansen
mesh_terms: D000327:Adsorption; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D012910:Snake Venoms / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D005227:Fatty Acids; D008246:Lysophospholipids; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010743:Phospholipids; D012910:Snake Venoms; D010741:Phospholipases A; C028694:1-palmitoyl-2-oleoylphosphatidylcholine