Asymmetry of membrane fluidity in the lipid bilayer of blood platelets: Fluorescence study with diphenylhexatriene and analogs

Abstract

Membrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37 degrees C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15 degrees C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.

Evidence for a lipochaperonin: Association of active proteinfolding GroESL oligomers with lipids can stabilize membranes under heat shock conditions

“…To assess the effect of GroEL 14 on the molecular order of lipid bilayers, fluorescence anisotropy measurements were carried out with LUVs labeled with the hydrophobic probes DPH or TMA-DPH. The fluorescence anisotropy of DPH embedded in lipid membranes is a measure of the motional order within the hydrophobic core of the membrane, while TMA-DPH reports movements in the headgroup region (33). Regardless of the probe position, GroEL 14 preincubated with either DMPC or DPPC LUVs had no effect on the order of the membrane below the temperature of the main gel-to-liquid-crystalline phase transition [DMPC T c ϭ 23ЊC; DPPC T c ϭ 41ЊC (34)].…”

Interaction of the Macrolide Antibiotic Azithromycin with Lipid Bilayers: Effect on Membrane Organization, Fluidity, and Permeability

“…Incorporation of fluorescent markers (at a molar ratio to the lipids of 1:250) was obtained by a vigorous mixing followed by a preincubation at 37°C during 1 h. Two markers with distinct localization in the lipid bilayer were used for comparison (diphenylhexatriene [DPH] a totally hydrophobic probe, which penetrates deeply into the membrane, and trimethylammoniumdiphenylhexatriene [TMA-DPH], which spans the hydrophilic/hydrophobic interface because of its amphiphilic character (21)(22)(23).…”

Rapid alterations in lateral mobility of lipids in the plasma membrane of activated human megakaryocytes

“…Studies based on fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, which detects the angular movement of a lipophilic molecule around a perpendicular axis in the plane of the membrane, showed a rigidification of the membrane after stimulation ionomycin had no effect [6]. 1 -(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene binds more specifically to the outer layer of the plasma membrane [6,71. Such labeled platelets again showed a decrease in membrane fluidity at low doses of thrombin (G0.2 U/ml) [8,91, but higher doses made the membrane more fluid [9].…”