Insights into sphingolipid miscibility: separate observation of sphingomyelin and ceramide N-acyl chain melting

Abstract

Ceramide produced from sphingomyelin in the plasma membrane is purported to affect signaling through changes in the membrane's physical properties. Thermal behavior of N-palmitoyl sphingomyelin (PSM) and N-palmitoyl ceramide (PCer) mixtures in excess water has been monitored by ²H NMR spectroscopy and compared to differential scanning calorimetry (DSC) data. The alternate use of either perdeuterated or proton-based N-acyl chain PSM and PCer in our ²H NMR studies has allowed the separate observation of gel-fluid transitions in each lipid in the presence of the other one, and this in turn has provided direct information on the lipids' miscibility over a wide temperature range. The results provide further evidence of the stabilization of the PSM gel state by PCer. Moreover, overlapping NMR and DSC data reveal that the DSC-signals parallel the melting of the major component (PSM) except at intermediate (20 and 30 mol %) fractions of PCer. In such cases, the DSC endotherm reports on the presumably highly cooperative melting of PCer. Up to at least 50 mol % PCer, PSM and PCer mix ideally in the liquid crystalline phase; in the gel phase, PCer becomes incorporated into PSM:PCer membranes with no evidence of pure solid PCer.

Figure 1

²H NMR spectra for 90:10 PSM-D31:PCer and 90:10 PSM:PCer-D31 at various temperatures. (Boxed temperatures) Boundaries of the phase transition. The minor baseline irregularities at ±60 kHz in the 56–75°C spectra of 90:10 PSM:PCer-D31 are artifacts from acoustic ringing.

Figure 2

Effect of PCer concentration on the temperature dependence of M₁ for 100:0(●), 90:10(▴), 80:20(▾), 70:30(♦), 60:40(■) PSM-D31:PCer (solid symbols), and PSM:PCer-D31 (open symbols).

Figure 3

Order parameter profiles for 90:10, 80:20, 70:30, and 60:40 PSM-D31:PCer (solid symbols) and PSM:PCer-D31 (open symbols) at 75°C. All membranes are in the L_α phase. S_CD values within the plateau regions are average values because the quadrupolar splittings from deuterons on these carbons are unresolved.

Figure 4

The negative integral of the DSC thermograms of PSM:PCer (dotted lines) are superimposed on ²H NMR M₁ temperature-dependence graphs for 100:0, 90:10, 80:20, 70:30, and 60:40 PSM-D31:PCer (solid symbols) and PSM:PCer-D31 (open symbols). The two graphs are scaled such that the maximum and minimum values of the integral and M₁ are aligned.

Figure 5

Phase diagram constructed from spectral inspections of PSM-D31:PCer (▴), spectral inspections of PSM:PCer-D31 (▵), spectral subtractions of PSM:PCer-D31 (○), completion of the highest-temperature DSC endotherm (■), and the apex temperature of the low-temperature endotherm (∼40°C) for 0–30% PCer and low-temperature onset of the broad endotherm (∼70°C) for 40 and 50% PCer on DSC thermograms (□).

Figure 6

Phase boundaries calculated from regular solution theory based on the assumption of ideal mixing (solid lines) superimposed on the experimental data of Fig. 5.

Figure 7

The PSM:PCer partial phase diagram. (Solid lines) Liquidus and the three-phase line at 41°C reflecting the good agreement of DSC and ²H NMR data. Other phase boundaries are expected, and we offer possible locations for these (dashed lines). An interpretation of the phases is also shown and corresponds to labeled regions of the phase diagram in Fig. 5. (Shaded and solid lipids) PSM and PCer, respectively. (Straight tails) Gel phase lipids. (Curvy tails) L_α phase lipids.