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Purified myelin membranes (PMMs) are the starting material for biochemical studies, from individual components up to the isolation of detergent-resistant membrane (DRM) fractions or detergent-insoluble glycosphingolipid (DIG) fractions, which are commonly believed to resemble physiological lipid rafts. The normal DIG isolation protocol involves the extraction of lipids under moderate cooling. The isolation of PMMs also involves the cooling of myelin as well as exposure to low ionic strength (IS). Here, we addressed the combined influence of cooling and IS on the structure of PMMs. The phase behaviour was investigated by small angle X-ray diffraction. Analysis of the diffraction peaks revealed the lamellar periodicity (d), the number of periodically correlated bilayers (N), and the relatives fractions of each phase. Departure from physiological conditions induced a phase separation in myelin. The effect of monovalent and divalent ions was also compared at equivalent IS, showing a differential effect, and phase diagrams for both ion types were established — Ca²⁺ induced the well-known over-compacted phase, but additionally we also found an expanded phase at low IS. Na⁺ promoted phase separation, and also induced over-compaction at sufficiently high IS. Finally, exploring the whole phase diagram, we found evidence for the direct isothermal transformation from the expanded to the compacted phase, suggesting that both phases could in fact originate from the identical primary lateral phase separation, whereas the apparent difference lies in the inter-bilayer interaction that is modulated by the ionic milieu.

Purified myelin membranes (PMMs) are the starting material for biochemical analyses such as the isolation of detergent-insoluble glycosphingolipid-rich domains (DIGs), which are believed to be representatives of functional lipid rafts. The normal DIGs isolation protocol involves the extraction of lipids under moderate cooling. Here, we thus address the influence of cooling on the structure of PMMs and its sub-fractions. Thermodynamic and structural aspects of periodic, multilamellar PMMs are examined between 4°C and 45°C and in various biologically relevant aqueous solutions. The phase behavior is investigated by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Complementary neutron diffraction (ND) experiments with solid-supported myelin multilayers confirm that the phase behavior is unaffected by planar confinement. SAXS and ND consistently show that multilamellar PMMs in pure water become heterogeneous when cooled by more than 10–15°C below physiological temperature, as during the DIGs isolation procedure. The heterogeneous state of PMMs is stabilized in physiological solution, where phase coexistence persists up to near the physiological temperature. This result supports the general view that membranes under physiological conditions are close to critical points for phase separation. In presence of elevated Ca²⁺ concentrations (> 10 mM), phase coexistence is found even far above physiological temperatures. The relative fractions of the two phases, and thus presumably also their compositions, are found to vary with temperature. Depending on the conditions, an “expanded” phase with larger lamellar period or a “compacted” phase with smaller lamellar period coexists with the native phase. Both expanded and compacted periods are also observed in DIGs under the respective conditions. The observed subtle temperature-dependence of the phase behavior of PMMs suggests that the composition of DIGs is sensitive to the details of the isolation