Abstract
We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous liquid-expanded (LE) SM-enriched phase. Langmuir-Schaefer films were made in order to visualize the labeled enzyme. Independently of initial conditions Alexa-SMase is preferably localized in the SM-enriched LE phase and it is not enriched at the domain boundaries. A novel mechanism is proposed for the action of SMase, which can also explain the regulatory effect of the surface topography on the enzyme activity. The homogeneous enzymatic generation of Cer in the LE phase leads to a meta-stable, kinetically trapped, supersaturated mixed monolayer. This effect acts as driving force for the segregation of the Cer-enriched domain following classical nucleation mechanisms. Accordingly, the number and size of Cer-enriched domains are determined by the extent of Cer supersaturation in the LE phase rather than by the SMase local activity. The kinetic barrier for nucleation, for which a compositional gap of at least 53 mol% of Cer is necessary to reach a thermodynamically stable LC phase, can explain the lag time to reaching full catalytic activity. Altogether, the data support an "area-activated mechanism," in which the enzyme is homogeneously active over the LE surface.
Highlights
We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous liquid-expanded (LE) SMenriched phase
More recent studies [12] have demonstrated that fluorescein-labeled phospholipase A2 (PLA2) from Crotalus atrox was homogeneously distributed in 1palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and DPPC giant unilamellar vesicles when the lipids were in the LE state and preferentially localized in the LE phase at temperatures corresponding to the gel-fluid phase coexistence
We found that SMase is preferably localized in the LE phase of the lipid monolayers
Summary
We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous liquid-expanded (LE) SMenriched phase. The homogeneous enzymatic generation of Cer in the LE phase leads to a meta-stable, kinetically trapped, supersaturated mixed monolayer This effect acts as driving force for the segregation of the Cer-enriched domain following classical nucleation mechanisms. In 1990, it was reported that the liquidexpanded (LE), liquid-condensed (LC) lateral interfaces (lipid domain borders) in a one-component monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) act as starting points for Naja naja phospholipase A2 (PLA2) catalytic activity [9]. Dahmen-Levison, Brezesinski, and Mohwald [10] showed in lipid monolayers that the fluorescent-labeled PLA2 preferably accumulates at the LC-LE interface of domains Those results support the socalled perimeter-activated or border-activated mechanism, in which the enzyme must have physical contact with the domain boundary/membrane defect in order to become fully active and exerts its major catalytic action adsorbed to these linear interfaces [11].
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