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

Specific adsorption of pla(2) at monolayers.

Specific phospholipase A2 (PLA2) adsorption studies were performed on monolayers of d-dipalmitoyl-phosphatidylcholine (d-DPPC) and of an ether-ester d-1-O-hexadecyl-2-stearoyl-phosphatidylcholine (d-HSPC). In order to separate interfacial recognition from subsequent lipid cleavage, PLA2-resistant d-enantiomers were utilized for the investigations. Snake venom (N. naja naja and Crotalos atrox) PLA2, which hydrolyzes the sn-2 ester bond of l-phospholipids, was used. Fluorescence microscopy, film-balance pressure–area isotherms, and grazing incidence X-ray diffraction (GIXD) experiments were carried out. Fluorescence microscopy studies show that the enzyme accumulates preferentially at the liquid-expanded/condensed interface. At low surface pressure enzyme penetration into the monolayer is observed, whereas at high pressures the area per molecule is reduced upon specific adsorption. Monolayer structure, as determined by GIXD, is greatly affected by adsorption of PLA2. The tilt angle of the aliphatic chains of the monolayer becomes drastically reduced due to an enzyme-induced increase of the lipid packing efficiency. The unspecific adsorption of serum albumin to a d-DPPC monolayer does not change the monolayer structure. The structural changes, caused by PLA2 adsorption on d-enantiomer monolayers, are related to the chemical structure of the lipid molecules. Therefore, a relation between structure change and hydrolysis efficiency of PLA2 on the respective l-enantiomer monolayers can be assumed

Direct observations of the cleavage-reaction of an L-DPPC monolayer catalyzed by phospholipase A(2) and inhibited by an indole inhibitor at the air/water interface

The enzymatic hydrolysis of an L-dipalmitoylphoisphatidylcholine (L-DPPC) monolayer at the air/water interface, catalyzed by phospholipase A(2) (PLA(2)), serves as a model for biospecific interfacial reactions. The cleavage of L-DPPC was investigated by Brewster angle microscopy. Different types of domain defects were observed to form in the coexisting liquid expanded and liquid condensed phases during the hydrolysis reaction. The adsorption of the enzyme was quantitatively recorded as the increase of the surface pressure over a fixed molecular area with time. In the case of the following processes: adsorption of PLA(2), cleavage reaction, and rearrangement of substrate and product molecules at the interface. Addition of a PLA(2) inhibitor to the lipid monolayer leads to a fast surface pressure increase after enzyme injection. The surface pressure reaches a maximum value and then does not change for a long time. During this period, no change in the domain shape and number density was observed, which indicates that the enzyme is inhibited for a certain period of time. The experimental results provide the possibility of a direct way to prove inhibitor activity.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000182220400007&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Biochemistry & Molecular BiologyChemistry, MedicinalSCI(E)14ARTICLE4299-305