Membrane fluidity analyses by general polarization and anisotropy.

Steady-state fluorescence measurements were performed on an LS 55 spectrofluorometer (PerkinElmer LAS GmbH, Rodgau, Germany) equipped with a pulsed xenon lamp, excitation and emission monochromators, and polarizing filters. The sample temperature was regulated with a Peltier element (PTP-1; PerkinElmer LAS GmbH, Rodgau, Germany). Two different probes were used to measure different physical properties of biomembranes. Laurdan GP was used to determine membrane order and TMA-DPH anisotropy to measure motility of the lipid molecules (37).

For Laurdan measurements, washed cells were suspended in 48 mM K₂HPO₄ (pH 7.4) buffer and diluted to an optical density at 625 nm (OD₆₂₅) of 0.2. According to the method of Molina-Höppner et al. (42), Laurdan stock solution was prepared in ethanol at 2 mM and stored in the dark at 4°C. Staining was performed at a concentration of 20 μM for 30 min at 30°C in the dark. Labeled cells were washed twice with particle-free 6°C precooled K₂HPO₄ buffer by centrifugation at 2,060 × g at 6°C for 10 min. Nonstained cells were used to determine blank values. Two milliliters of suspension was transferred to a 3,500-μl quartz cuvette (Hellma GmbH, Müllheim, Germany). Emission spectra (slit, 3.0 nm) were recorded from 380 nm to 600 nm following excitation (slit, 10.0 nm) at 360 nm at the indicated temperatures. GP values were calculated using emission values (I) at 435 nm and 500 nm (37) as follows: GP = (I₄₃₅ − I₅₀₀)/(I₄₃₅ + I₅₀₀).

TMA-DPH staining was done analogously to Laurdan staining and as previously described by Usui et al. (43) and Abe and Hiraki (44). Anisotropy measurements of amino acid-supplemented cells were done in KCl solution (pH 7.0) instead of phosphate buffer. TMA-DPH stock solution was prepared in dimethyl sulfoxide (DMSO) at a concentration of 400 μM. The cells were stained with 0.5 μM TMA-DPH for 10 min at 30°C in the dark and washed twice. The excitation and emission wavelengths were 355 nm and 425 nm, respectively. Anisotropy values were calculated from polarized intensities using the following equation: r = (I_VV − GI_VH)/(I_VV + 2GI_VH), where I is the fluorescence intensity from which blank values from nonlabeled cells were subtracted. G stands for G factor, calculated as the ratio I_HV/I_HH. H (horizontal) und V (vertical) indicate the polarizer positions for the excited and the emitted light. Each data point was calculated from 10 to 20 single measurements. The data are shown as means with standard deviations from independent biological triplicates.

The fluidizing effect of naphthoquinones on biomembranes was confirmed in defined MLVs with increasing quinone supplementation. Vesicles were formed according to the protocol of Harris et al. (37). DPPC was purchased from Avanti Polar Lipids (850355; Alabaster, AL) and TMA-DPH and vitamin K₁ from Sigma-Aldrich. MLVs were assembled without vitamin K₁ supplementation and with 1 and 10% vitamin K₁ supplementation.