Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 1. Single component phosphatidylcholine liposomes

Lentz, Barry R.; Barenholz, Y.; Thompson, T. E.

doi:10.1021/bi00665a029

Cited by 472 publications

(265 citation statements)

References 18 publications

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“…Furthermore, these studies show that the average environment of NBD-PE, as monito by the absolute position remains unaffected by these changes. These results are not particularly surprising in view of the fact that changes in membrane organization brought about by phase transition (or temperature) are largely restricted to the fatty acyl region of the membrane (Lentz et al, 1976) and are not sensed by the NBD moiety, which is linked to the headgroup in the case of NBD-PE and is located at the membrape interface (Chattopadhyay & London, 1987Paejano & Martin, 1988;Chattopadhyay, 1990;Mitra & Hammes, 1990;Wolf et al, 1992). It has been previously reported that the rates of solvent relaxation for probes that are not qeeply buried in the hydrocarbon interior of the membrane are more or less independent of temperature (Lakowicz et al, 1983a;takowicz & Keating-Nakamoto, 1984).…”

Section: 'Vv Crlvhmentioning

confidence: 94%

Fluorophore environments in membrane-bound probes: A red edge excitation shift study

Chattopadhyay

Mukherjee²

1993

Biochemistry

140

158

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A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the Red Edge Excitation Shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. In this paper, we report the red edge excitation shift of a membrane-bound phospholipid molecule whose headgroup is covalently labeled with a 7-nitrobenz-2-oxa-1,3-diazol-4-y1 (NBD) moiety. When incorporated into model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), the NBD-labeled phospholipid (NBD-PE), exhibits a red edge excitation shift of 10 nm. In addition, fluorescence polarization of NBD-PE in membranes shows both excitation and emission wavelength dependence. The nonpolar membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) does not show red edge excitation shift in model membranes. The lifetime of NBD-PE in DOPC vesicles was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD moiety in the membrane. The magnitude of the red shift in the emission maximum for NBD-PE was found to be independent of temperature, between 12 and 54 O C , and of the physical state (gel or fluid) of the membrane. Taken together, these observations are indicative of the motional restriction experienced by this fluorophore in the membrane. Red edge excitation shift promises to be a powerful tool in probing membrane organization and dynamics.Organized molecular assemblies such as membranes can be considered as large cooperative units with characteristics very different from the individual structural units which constitute them. Fluorescence spectroscopy has been one of the principal techniques to study organization and dynamics of biological and model membranes because of its suitable time scale, noninvasive nature, and intrinsic sensitivity (Radda, 1975;Lakowicz, 1980Lakowicz, , 1981. For a fluorophore in a bulk nonviscous solvent, the dipolar relaxation of the solvent molecules around the fluorophore in the excited state is much faster than the fluorescence lifetime. The fluorescence decay rates and the wavelength of maximum emission, in such a case, are therefore usually independent of the excitation wavelength. However, these parameters do show excitation wavelength dependence if the dipolar relaxation of the solvent molecules is slow in the excited state, such that the relaxation time is comparable to or longer than the fluorescence lifetime (Chen, 1967;Fletcher, 1968;Galley & Purkey, 1970;Rubinov & Tomin, 1970;Castelli & Forster, 1973;Itoh & Azumi, 1975;Demchenko, 1982; Lakowicz & Keating-Nakamoto, 1984). Such a shift in the wavelength of maximum emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitatio...

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Section: 'Vv Crlvhmentioning

confidence: 94%

Fluorophore environments in membrane-bound probes: A red edge excitation shift study

Chattopadhyay

Mukherjee²

1993

Biochemistry

140

158

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“…Structural Studies-Phospholipid acyl chain and head group packing order was determined by labeling rHDL with 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) (32,33), respectively. Steady state fluorescence polarization of the DPH-and TMA-DPHlabeled rHDL was measured at 5°C intervals from 5 to 50°C using an excitation wavelength of 366 nm.…”

mentioning

confidence: 99%

The Mechanism of the Remodeling of High Density Lipoproteins by Phospholipid Transfer Protein

Settasatian¹,

Duong²,

Curtiss³

et al. 2001

Journal of Biological Chemistry

114

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Phospholipid transfer protein (PLTP) remodels high density lipoproteins (HDL) into large and small particles. It also mediates the dissociation of lipid-poor or lipid-free apolipoprotein A-I (apoA-I) from HDL. Remodeling is enhanced markedly in triglyceride (TG)-enriched HDL (Rye, K.-A., Jauhiainen, M., Barter, P. J., and Ehnholm. C. (1998) J. Lipid. Res. 39, 613-622). This study defines the mechanism of the remodeling of HDL by PLTP and determines why it is enhanced in TG-enriched HDL. Homogeneous populations of spherical reconstituted HDL (rHDL) containing apoA-I and either cholesteryl esters only (CE-rHDL; diameter 9.3 nm) or CE and TG in their core (TG-rHDL; diameter 9.5 nm) were used. After 24 h of incubation with PLTP, all of the TG-rHDL, but only a proportion of the CE-rHDL, were converted into large (11.3-nm diameter) and small (7.7-nm diameter) particles. Only small particles were formed during the first 6 h of incubation of CE-rHDL with PLTP. The large particles and dissociated apoA-I were apparent after 12 h. In the case of TG-rHDL, small particles appeared after 1 h of incubation, while dissociated apoA-I and large particles were apparent at 3 h. The composition of the large particles indicated that they were derived from a fusion product. Spectroscopic studies indicated that the apoA-I in TG-rHDL was less stable than the apoA-I in CE-rHDL. In conclusion, these results show that (i) PLTP mediates rHDL fusion, (ii) the fusion product rearranges by two independent processes into small and large particles, and (iii) the more rapid remodeling of TG-rHDL by PLTP may be due to the destabilization of apoA-I. Phospholipid transfer protein (PLTP)1 transfers phospholipids (PL) between high density lipoproteins (HDL) and very low density lipoproteins as well as between different particles within the HDL fraction (1, 2). It also remodels HDL into large and small particles in a process that is accompanied by the dissociation of lipid-poor or lipid-free apolipoprotein A-I (apoA-I) (3-8). Remodeling is enhanced markedly in HDL that contain triglyceride (TG) in their core (9). Evidence of the importance of PLTP in HDL metabolism comes from studies of mice transgenic for human PLTP. These animals have increased levels of pre-␤ 1 -migrating HDL, the initial acceptors of cellular cholesterol in the first step of the reverse cholesterol pathway, and are also resistant to intracellular cholesterol accumulation (10 -12). Studies of PLTP knockout mice have shown that PLTP is essential for maintaining normal HDL levels in plasma (13). Moreover, it has been reported recently that PLTP-mediated transfers of phospholipids between HDL and other lipoprotein classes are not interchangeable with the phospholipid transfers that are mediated by cholesteryl ester transfer protein (CETP) (14).The mechanism of the remodeling of HDL by PLTP is poorly understood. Although there is evidence that particle fusion and the dissociation of lipid-poor or lipid-free apoA-I are involved (7,8), nothing is known about how the interaction of PLTP wi...

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“…Thus, the kinetics of transport processes are more complex due to multiple diffusion barriers and it is not easy to obtain a uniform-sized population of vesicles. These disadvantages are overcome by the use of small, unilamellar vesicles; however, the surface of these vesicles has a high degree of curvature which affects many of the physical properties of the phospholipid in the vesicles (8,9). One would then expect the large, unilamellar vesicles to resemble most closely biological membranes.…”

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confidence: 99%

Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.

Enoch¹,

Strittmatter²

1979

Proc. Natl. Acad. Sci. U.S.A.

215

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Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. (5) or ether injection (6). Vesicles may be formed with phospholipid mixtures, purified phospholipids, or synthetic phospholipids. The methods of preparation and the properties of such vesicles have recently been reviewed (1, 7). Although multilamellar liposomes are easy to prepare, they have been criticized as a model system for the study of membrane phenomena (1). Thus, the kinetics of transport processes are more complex due to multiple diffusion barriers and it is not easy to obtain a uniform-sized population of vesicles. These disadvantages are overcome by the use of small, unilamellar vesicles; however, the surface of these vesicles has a high degree of curvature which affects many of the physical properties of the phospholipid in the vesicles (8, 9). One would then expect the large, unilamellar vesicles to resemble most closely biological membranes. It was previously shown (4) that treatment of 1 mol of egg lecithin with 2 mol of bile salt followed by gel filtration to remove the bile salt results in the formation of a uniform population of single-bilayer vesicles having an average diameter of 300 A. We now report that the treatment of 1 mol of egg lecithin with 0.5 mol of bile salt results in the formation of a uniform population of single-bilayer vesicles having an average diameter of 1000 A. These preparations, which have a large internal volume and are free of multilamellar structures, may be useful in cases in which previous techniques (5, 6) cannot be applied.MATERIALS AND METHODS Egg phosphatidylcholine (10), sodium deoxycholate (11), cytochrome b5 heme peptide (12), the catalytic fragment of NADH-cytochrome b5 reductase (13) (pH 5) or 1% ammonium molybdate (pH 5). One drop of liposomes (30-100 ,uM phospholipid) was placed on a Formvar-coated grid and, after 30 sec, one drop of staining solution was added; 30 sec later, the solution was drained off with filter paper and the grid was allowed to air dry. The grids were examined in a Hitachi llE electron microscope operated at 75 kV. Vesicle diameters were measured on several electron micrographs covering different areas on a grid and then averaged from at least 400 measurements.Method I: Formation of Large Vesicles from Small, Preformed Vesicles. Small, uniform-sized egg lecithin vesicles were prepared as described (11). Any solute to be entrapped was added to the vesicle solution at this point and the vesicles were adjusted to 20 mM phospholipid. The solution was then warmed to 25°C and an aliquot of 250 mM sodium deoxycholate was rapidly added and mixed to give a final mixture containing a ratio of deoxycholate to phospholipid of 1:2. The large vesicles began to form almost immediately, as indicated by. the increase in the light scattering of the solution, a change from nearly clear for the small vesicles to a transparent opalescence for the large vesicles. Vesicle formation was complete within 5-10 min at 250C (vesicles could also be formed at 40C...

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Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 1. Single component phosphatidylcholine liposomes

Cited by 472 publications

References 18 publications

Fluorophore environments in membrane-bound probes: A red edge excitation shift study

Fluorophore environments in membrane-bound probes: A red edge excitation shift study

The Mechanism of the Remodeling of High Density Lipoproteins by Phospholipid Transfer Protein

Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.

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