Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A(2) Action at the Single Molecule Level
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Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A(2) Action at the Single Molecule Level. / Gudmand, Martin Jesper; Rocha, Susana ; Hatzakis, Nikos; Peneva, Kalina ; Müllen , Klaus ; Stamou, Dimitrios; Uji-I, Hiroshi; Hofkens , Johan ; Bjørnholm, Thomas; Heimburg, Thomas Rainer.
In: Biophysical Journal, Vol. 98, No. 9, 05.05.2010, p. 1873-82.Research output: Contribution to journal › Journal article › Research › peer-review
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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 A(2) (PLA(2)) 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 PLA(2) 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 PLA(2). 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 microm(2)/s on the L-DPPC and 4.9 microm(2)/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to approximately 0.2 microm(2)/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 approximately 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 A(2) (PLA(2)) 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 PLA(2) 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 PLA(2). 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 microm(2)/s on the L-DPPC and 4.9 microm(2)/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to approximately 0.2 microm(2)/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 approximately 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
VL - 98
SP - 1873
EP - 1882
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 9
ER -
ID: 33239624