Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures

Abstract

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

“…All curves shown in Figure 1 are uncorrected for artifacts due to light scattering. We have shown earlier (Martin & Thompson, 1978) that the low optical density of lipid samples precludes the need for corrections, but the much higher turbidity of the membrane suspensions creates potential problems. When experimental data are collected by using polarized light, any scattering of either the excitation or the emission photons can result in an apparent decrease in polarization by changing the orientation of the measured light (Teale, 1969;Lentz et al, 1979) .…”

“…Fluorescence Measurements. Lipid multibilayer vesicles were prepared from microsomal lipids or synthetic lipids as described earlier (Martin & Thompson, 1978). Diphenylhexatriene was dissolved in organic solvents containing lipids to give a probe to lipid molar ratio of 1:500.…”

“…Steady-state polarization measurements were made with the custom-made device described by Martin & Thompson (1978), modified by interfacing with a PET 2001 series computer (Commodore, Palo Alto, CA) as previously described (Dickens et al, 1980). Temperature regulation was with a jacketed cuvette and a circulating water bath as previously described (Dickens et al, 1980).…”

Simplified derivation of angular order and dynamics of rodlike fluorophores in models and membranes. Simultaneous estimation of the order and fluidity parameters for diphenylhexatriene by only coupling steady-state illumination polarization and lifetime of fluorescence

“…Now, comparisons between the values of 1/(4) given by previous authors in membranes (2,(39)(40)(41)(42)(43), and those we obtained by our method (from the S values given by the same authors) become possible. Some of them are shown in Fig.…”

Genetic Engineering of Osmoregulation

“…Tetrahymena does not, however, maintain a precisely constant viscosity. Experiments with 5-nitroxide stearate spin label showed that the order parameter of cells grown at 34 C was 87% of that of cells grown at 15 C. The membranes of the low-temperature cells are thus more viscous than those of the high-temperature cells (Nozawa et al, 1974; see also Martin and Thompson, 1978;Martin and Foyt, 1978).…”