Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A(2) Action at the Single Molecule Level

TY - JOUR

T1 - Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A(2) Action at the Single Molecule Level

AU - Gudmand, Martin Jesper

AU - Rocha, Susana

AU - Hatzakis, Nikos

AU - Peneva, Kalina

AU - Müllen , Klaus

AU - Stamou, Dimitrios

AU - Uji-I, Hiroshi

AU - Hofkens , Johan

AU - Bjørnholm, Thomas

AU - Heimburg, Thomas Rainer

N1 - Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

PY - 2010/5/5

Y1 - 2010/5/5

N2 - We monitored the action of phospholipase A2 (PLA2) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm2/s on the L-DPPC and 4.9 μm 2/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm2/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.

AB - We monitored the action of phospholipase A2 (PLA2) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm2/s on the L-DPPC and 4.9 μm 2/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm2/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.

KW - 1,2-Dipalmitoylphosphatidylcholine

KW - Animals

KW - Diffusion

KW - Fluorescent Dyes

KW - Imides

KW - Perylene

KW - Phospholipases A2

KW - Solubility

KW - Stereoisomerism

KW - Substrate Specificity

KW - Water

U2 - 10.1016/j.bpj.2010.01.035

DO - 10.1016/j.bpj.2010.01.035

M3 - Journal article

C2 - 20441751

SN - 0006-3495

VL - 98

SP - 1873

EP - 1882

JO - Biophysical Journal

JF - Biophysical Journal

IS - 9

ER -