Assays for transmembrane movement of sphingolipids

This chapter compares the available techniques to study the transmembrane distribution of sphingolipids, and describe the requirements that these assays should meet in order to be applicable to measurements of transbilayer mobility. Translocation activity of a particular protein can then be tested by transfection into a cellular assay system, or by reconstitution into model membranes. To determine transmembrane movement of a sphingolipid, an assay first must be developed to measure its distribution across the bilayer at a specific moment in time. The following criteria can be applied to judge the usefulness of a particular approach: (1) The procedure must recognize the lipid of interest on a membrane surface, (2) The procedure must discriminate the lipids in the one leaflet from those in the opposite leaflet of the bilayer, (3) The amount of the lipid under study should not change during the assay due to processes independent of the assay, such as via uptake by exocytosis and endocytosis with hydrolysis and (re)synthesis. Lipid sidedness and translocation may also be studied by using analogs of the lipids of interest, Examples are fluorescent analogs, spin-labeled analogs, analogs with shortened acyl chains, or a combination of these. Their sideness may then be monitored in time by the techniques mentioned above or by noninvasive spectroscopic methods. Generally, results are more convincing when confirmed by independent methods.