Sphingomyelinase D Activity in Model Membranes: Structural Effects of in situ Generation of Ceramide-1-Phosphate

R.P. STOCK; J. BREWER; K. WAGNER; B. RAMOS-CERRILLO; L. DUELUND; KIT DRESCHER JERNSHØJ; L. FOLKE-OLSEN; LUIS A. BAGATOLLI

PLOS ONE

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2012

1932-6203

he toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) cer