Submitted on March 7, 2008
Accepted on May 27, 2008
Sphingomyelinase acts by an area-activated mechanism on the liquid-expanded phase of sphingomyelin monolayers
Luisina De Tullio, Bruno Maggio, and María Laura Fanani
Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Córdoba X5000HUA
Corresponding Author: lfanani{at}gmail.com
We described the localization of Alexa-488-labeled Sphingomyelinase (SMase) in sphingomyelin (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 labelled 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 segregation of 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 53mol% of Cer is necessary to reach a thermodynamically stable LC phase, can explain the lag time for reach full catalytic activity. All together the data support an “area-activated mechanism” where the enzyme is homogeneously active over the LE surface.
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