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note
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Stained samples for transmission electron microscopy were prepared by drying the suspension on a carbon-coated Formvar grid and then negatively staining with 1% uranyl acetate. Freeze-fracture samples were rapidly frozen by slamming them against a copper plate at -150 °C (Gentleman Jim). Fracture and deep etching were performed on a Cressington CFE 40. Electron microscopy was performed on a Zeiss EM-10C or a JEOL 120 CX. Circular dichroism studies were carried out on a Jasco J-720 spectropolarimeter. Samples were removed from the refrigerator and immediately placed in a water-jacketed quartz cell (0.5 mm path length) that had been precooled to 4 °C by a water circulator (Neslab). Spectra were recorded every 0.5 °C while heating at a rate of 4 °C/hour. A 400 MHz, Bruker DRX-400 spectrometer was used to record the NMR spectra. The ratio of lipids was determined by comparing the number of terminal methyl protons (δ0.88 ppm, triplet) from both lipids to the total number of methylene protons in the a-position to the diacetylenic group (δ2.27 ppm, multiplet). In an equimolar mixture, this ratio should be 12:16.
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