Lag-burst kinetics in phospholipase A(2) hydrolysis of DPPC bilayers visualized by atomic force microscopy.

The lag-burst phenomenon in the phospholipase A(2) mediated hydrolysis of phospholipid bilayers is for the first time demonstrated in an atomic force microscopy (AFM) study. Simultaneous AFM measurements of the degree of bilayer degradation and the physical-chemical state of the membrane reveals growing nanoscale indentations in the membrane during the lag phase. It is argued that these indentations are domains of hydrolysis products (lysoPC/PC) which eventually trigger the burst. The rate of the rapid hydrolysis following the burst is found to be proportional to the length of the edge between membrane adsorbed and desorbed to the mica base. The observed maximal rate of membrane degradation is approx. 0.2 mmol lipid/min/mol lipase in solution.

Increase in phospholipase A2 activity towards lipopolymer-containing liposomes.

Phospholipase A2 (PLA2)-catalyzed hydrolysis of dipalmitoylphosphatidylcholine (DPPC) liposomes incorporated with submicellar concentrations of polyethyleneoxide covalently attached to dipalmitoylphosphatidylethanolamine (DPPE-PEG2000) has been studied in the gel-to-fluid transition region of the host DPPC lipid bilayer matrix. By means of fluorescence and light-scattering measurements, the characteristic PLA2 lag time has been determined as a function of lipopolymer concentration and temperature. The degree of lipid hydrolysis was followed using radioactive labeled lipids. Differential scanning calorimetry has been applied to characterize the thermodynamic phase behavior of the lipopolymer-containing liposomes. A remarkable lipopolymer concentration-dependent decrease in the lag time was observed over broad temperature ranges. The radioactive measurements demonstrate an increase in catalytic activity for increasing amounts of lipopolymers in the bilayer. Hence, the lipopolymers act as a promoter of PLA2 lipid hydrolysis resulting in a degradation of the bilayer structure and a concomitant destabilization of the liposomes. This behavior is in contrast to the generally observed protective and stabilization effect in biological fluids exerted by lipopolymers in polymer-grafted liposomes. It is proposed that the enhanced activity of the small water soluble and interfacially active enzyme may involve a non-uniform distribution of the lipopolymers in the lipid matrix due to a coupling between local lipid bilayer curvature and composition of the non-bilayer-preferring lipopolymers.

Direct imaging by cryo-TEM shows membrane break-up by phospholipase A2 enzymatic activity.

Phospholipid hydrolysis to free fatty acid and 1-lyso-phospholipid by water-soluble phospholipase A2 (PLA2) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis. Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles. This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA2-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA2 activity and the morphology of the lipid suspension.