10 research outputs found
Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level
We monitored the action of phospholipase A2 (PLA2) on L- and
D-dipalmitoylphosphatidylcholine (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 build-up 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 (SPT). Diffusion coefficients of enzymes adsorbed to the
fluid interface were between 3 mu m^2/s on the L-DPPC and 4.6 mu m^/s on the
D-DPPC monolayers. In regions enriched with hydrolysis products the diffusion
dropped to approx. 0.2 mu m^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 approx. 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.Comment: 10 pages, 10 figure