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High-melting lipid mixtures and the origin of detergent-resistant membranes studied with temperature-solubilization diagrams.

Biophysical journal (2014-12-18)
Jesús Sot, Marco M Manni, Ana R Viguera, Verónica Castañeda, Ainara Cano, Cristina Alonso, David Gil, Mikel Valle, Alicia Alonso, Félix M Goñi
ABSTRACT

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4-50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24-48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cholesterol, Sigma Grade, ≥99%
Sigma-Aldrich
Cholesterol, powder, BioReagent, suitable for cell culture, ≥99%
Sigma-Aldrich
SyntheChol® NS0 Supplement, 500 ×, synthetic cholesterol, animal component-free, aqueous solution, sterile-filtered, suitable for cell culture
SAFC
Cholesterol, Plant-Derived
Sigma-Aldrich
Cholesterol, from sheep wool, ≥92.5% (GC), powder
Supelco
Cholesterol, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Cholesterol solution, certified reference material, 10 mg/mL in chloroform
Sigma-Aldrich
Di(N-succinimidyl) glutarate, ≥97.0% (CHN)
Sigma-Aldrich
1,2-Dipalmitoyl-sn-glycerol, ≥99%
SAFC
Cholesterol, from sheep wool, Controlled origin, meets USP/NF testing specifications