Methods to Characterize Synthesis and Degradation of Sphingomyelin at the Plasma Membrane and Its Impact on Lipid Raft Dynamics.

This chapter will discuss methods for analyses of the rates of sphingomyelin synthesis and turnover associated with lipid rafts or plasma membrane. These methods involve the use of fluorescently (NBD-C6-ceramide or NBD-C6-Sphingomyelin)) or radioactively labeled substrates ([H-methyl]-phosphatidylcholine, [H-acyl]-ceramide, [C-methyl]-sphingomyelin) to quantify in vitro the activity of the sphingomyelin synthase (SMS) (also known as phosphatidylcholine:ceramide phosphocholine transferase), acid sphingomyelinase (the endosomal/lysosomal (L-SMase) and the secretory (S-SMase) forms) and neutral sphingomyelinase-2 (nSMase-2). These methods allow to quantify changes in the activity of enzymes that affect the SM-to-ceramide ratio on the plasma membrane, and consequently, the lipid rafts biophysical properties, dynamics, and raft-associated receptor clustering and signaling events. Specific attention is paid to challenges caused by the fact that SMS and nSMase-2 are integral/membrane bound proteins and how to avoid the use of detergent that suppress their specific activities.