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Journal of Lipid Research, Vol. 49, 2347-2355, November 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Sphingomyelinase acts by an area-activated mechanism on the liquid-expanded phase of sphingomyelin monolayers

Luisina De Tullio, Bruno Maggio and María Laura Fanani¹

Centro de Investigaciones en Química Biológica de Córdoba, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA, Córdoba, República Argentina

Published, JLR Papers in Press, May 28, 2008.

This work was supported by Secretaría de Ciencia y Tecnología- Universidad Nacional de Córdoba (SECyT-UNC), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Fondo para la Investigación Científica y Tecnológica (FONCyT Argentina). B.M. and M.L.F. are career investigators of CONICET. L.D. is a doctoral fellow of CONICET.

¹ To whom correspondence should be addressed. e-mail: lfanani{at}gmail.com

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.

Supplementary key words lipid phase coexistence • fluorescent-labeled sphingomyelinase • domain nucleation • ceramide • epifluorescence microscopy • lateral segregation • Langmuir-Schaefer films

Abbreviations: Cer, ceramide; DiIC₁₂, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; LC, liquid-condensed; LE, liquid-expanded; PLA₂, phospholipase A₂; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; X_Cer, mole fraction of ceramide

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