Sphingosine-mediated membrane association of DNA and its reversal by phosphatidic acid

Abstract

Resonance energy transfer was measured between egg phosphatidylcholine liposomes containing the intramolecular excimer forming pyrene-labelled phospholipid analogue 1,2-bis[pyren-1-(-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a donor and DNA-bound adriamycin as an acceptor. Membrane association of DNA turned out to be critically dependent on the presence of sphingosine in the liposomes. Identical result was obtained by measuring the extent of quenching of the fluorescent DNA-bound dye Hoechst 33258 due to energy transfer to the lipophilic stain Nile Red incorporated in egg phosphatidylcholine liposomes containing varying amounts of sphingosine. The attachment of DNA to sphingosine-containing membranes could be reversed by the further inclusion of the negatively charged phosphatidic acid up to approximately 1:2 PA/sphingosine molar ratio in the liposomes, thus suggesting the involvement of electrostatic interactions. Differential scanning calorimetry measurements confirmed a lack of association between DNA and dimyristoylphosphatidylcholine liposomes. Instead drastic changes were produced by DNA in the heat capacity scans measured for liposomes also incorporating sphingosine. Fluorescence microscopy revealed an extensive aggregation of sphingosine containing pyrene-phosphatidylcholine-labelled egg phosphatidylcholine liposomes in the presence of DNA. Together with other available data on the effects of sphingosine, the present findings suggest that sphingosine could directly alter the chromatin structure. Accordingly, such alterations may contribute to the control of replication and gene expression.