Sonicated Phosphatidylcholine Vesicles Research Articles

Characteristics of bile acid-mediated Ca2+ release from permeabilized liver cells and liver microsomes

Saponin-treated liver cells and a microsomal fraction were used to characterize the mechanism of the Ca2+ release induced by different bile acids. The saponin-treated cells accumulated 0.8-1 nmol/mg of protein of the medium Ca2+ in a nonmitochondrial, high affinity, and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool. Three of five bile acids tested, lithocholate and the conjugates taurolithocholate and taurolithocholate sulfate, released 85% of the Ca2+ pool within 45-60 s and with ED50 from 16 to 28 microM. Ins(1,4,5)P3 released 80% from the same Ca2+ pool with an ED50 of 0.3 microM. The Ca2+-Mg2+-ATPase inhibitor vanadate (1 mM) had no effect on the Ca2+ released by the bile acids and Ins(1,4,5)P3. The Ins(1,4,5)P3-binding antibiotic neomycin (1 mM) and the receptor competitor heparin (16 micrograms/ml) abolished the releasing effect of Ins(1,4,5)P3 but had no effect on the bile acid-mediated Ca2+ release. The 45Ca2+ accumulated by the microsomal fraction (8 nmol of 45Ca2+/mg of protein) was released by the bile acids within 45-90 s and with an ED50 of 17 microM. In contrast, the bile acids had no effect on the Ca2+ permeability of other natural and artificial membranes. The resting 45Ca2+ influx of intact cells (0.45 nmol/mg of protein/min), the 45Ca2+ accumulated by mitochondria (2-13 nmol of 45Ca2+/mg of protein), and the 45Ca2+ trapped in sonicated phosphatidylcholine vesicles (5 mM 45Ca2+) were not altered by the different bile acids. These results suggest that the Ca2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool. It is not mediated by Ins(1,4,5)P3 or via activation of the Ins(1,4,5)P3 receptor, and it is specific for the membrane of the internal pool.

The effect of free cholesterol on the solubilization of cholesteryl oleate in phosphatidylcholine bilayers: A 13C-NMR study

The solubilization of cholesteryl oleate in sonicated phosphatidylcholine vesicles containing between 0 and 50 mol% cholesterol was studied by 13C-NMR using isotopically enriched [ carbonyl- 13C]cholesteryl oleate. The carbonyl- 13C chemical shift from cholesteryl oleate in the phospholipid/cholesterol bilayer was significantly downfield from that for cholesteryl oleate in an oil phase and the peak area, relative to that of the phospholipid carbonyl, was used to determine bilayer solubility of the ester. The ester solubility (with respect to phospholipid) in the phospholipid bilayer without cholesterol (2.9 mol%) was only moderately reduced (to 2.3 mol%) at cholesterol levels up to 33 mol% but showed a more marked reduction to 1.4 mol% at 40 mol% cholesterol or 1.2 mol% at 50 mol% cholesterol. Since the vesicles containing 50 mol% cholesterol were larger ( 520±152 A ̊ diameter ) than those with no cholesterol ( 291±97 A ̊ diameter ), we measured the solubility of cholesteryl oleate in large vesicles with no cholesterol, prepared by extrusion through polycarbonate membrane filters, and found it similar to that in small, sonicated vesicles with no cholesterol. Therefore, the larger size of vesicles was not the factor responsible for the decreased cholesteryl oleate solubility at high cholesterol contents. A more direct effect of cholesterol is envisioned where the ester becomes displaced to deeper regions of the bilayer.

Distribution of cytochrome b5 between small and large unilamellar phospholipid vesicles.

Cytochrome b5 is an amphipathic integral membrane protein that spontaneously inserts, post-translationally, into intracellular membranes. When added to preformed phospholipid vesicles, it binds in a so-called "loose" or transferable configuration, characterized by the ability of the protein to rapidly equilibrate between vesicles. In a preliminary report we showed that the distribution of cytochrome b5 among a heterogeneous population of small sonicated phosphatidylcholine vesicles (212 to about 350 A in diameter) lies in favor of the smallest vesicles by a factor of at least 20 (Greenhut, S.F. and Roseman, M.A. (1985) J. Biol. Chem. 260, 5883-5886). In the present studies we have attempted to determine the maximal extent to which bilayer curvature can influence the intervesicle distribution of cytochrome b5, by measuring the distribution of the protein between a population of limit-size vesicles 212 A in diameter and a population of large unilamellar vesicles approximately 1000 A in diameter. (The effect of bilayer curvature on the physical properties of the lipids in the large vesicles is considered to be negligible.) The results show that cytochrome b5 favors the small vesicle population by a factor of about 200. This observation suggests that the formation of highly curved regions in biological membranes (or the formation of regions in which the physical state of the lipids is similar to that in small vesicles) may cause the accumulation of certain membrane proteins at those sites. We also observed that a significant fraction (11-20%) of the cytochrome b5, when added directly to the large vesicles, spontaneously inserts into the "tight," physiologically proper configuration. A possible mechanism is discussed.

Distribution of cytochrome b5 between sonicated phospholipid vesicles of different size.

Cytochrome b5 is an amphipathic integral membrane protein that spontaneously inserts, post-translationally, into intracellular membranes. When added to preformed phospholipid vesicles, it binds in a so-called "loose," or transferable, configuration characterized by the ability of the protein to rapidly equilibrate between vesicles. A heterogeneous dispersion of sonicated phosphatidylcholine vesicles, 212 to about 350 A in diameter, was prepared by differential centrifugation. When cytochrome b5 was incubated with these vesicles (1 mol of protein/833 mol of phospholipid, in 0.01 M NaHCO3, 0.1 M NaCl, 10(-4) M EDTA, pH 7.4) and the mixture was subjected to molecular sieve chromatography on Sepharose 2B-CL, the cytochrome b5 was found to elute preferentially with the smaller vesicles. Subsequently, a fresh preparation of heterogeneous vesicles was subfractionated by gel filtration, and the individual fractions were incubated with the protein. Molecular sieve chromatography of these complexes showed that cytochrome b5 favors the smallest over the largest vesicles by a factor of at least 20. This result suggests that formation of highly curved regions in biological membranes may cause accumulation of certain membrane proteins at those sites.

Accelerated transfer of neutral glycosphingolipids and ganglioside GM1 by a purified lipid transfer protein.

The transfer of labeled neutral glycosphingolipids from sonicated phosphatidylcholine vesicles to erythrocyte ghosts is greatly stimulated by a nonspecific lipid transfer protein purified from beef liver. Globo-tetraglycosylceramide is transferred at a rate 40% of that for dipalmitoylphosphatidylcholine. II3-alpha-N-Acetylneuraminosyl-gangliotetraglycosylceramide is also transferred by the transfer protein, either from sonicated phosphatidylcholine vesicles or from ganglioside micelles to erythrocyte ghosts. The nonspecific lipid transfer protein catalyzes the net transfer of glycosphingolipids from brush border membrane vesicles (from rabbit intestine) to sonicated phosphatidylcholine/cholesterol vesicles.

Transbilayer movement of 24Na in sonicated phosphatidylcholine vesicles exposed to frequency-modulated microwave radiation.

Sonicated egg phosphatidylcholine vesicles loaded with 24Na+ were exposed at 20mW to a frequency-modulated (3 Hz) microwave field in the range of 2350 to 2550 MHz, or at 80 mW to a 2450-MHz CW (continuous wave) field, in a waveguide. The vesicle suspension absorbed microwaves at about 1 mW/ml and 25 mW/ml (CW experiment). The average temperature change of the irradiated suspension was less than 0.1 degree C from ambient. Leakage of 24Na+ from the vesicles for up to 19 hours was measured. No difference was noted in the movement of 24Na+ from the vesicles in the irradiated and control dispersions.

Glycoprotein micelles isolated from vesicular stomatitis virus spontaneously partition into sonicated phosphatidylcholine vesicles

Micelles formed by glycoprotein of vesicular stomatitis virus, isolated by octylglucoside extraction and purified free of detergent and phospholipid, spontaneously partitioned into preformed sonicated egg phosphatidylcholine vesicles when the glycoprotein rosettes and vesicles were incubated together in 10 m M Tris (pH 7.5) at 37°. The hydrophobic tail fragment of the glycoprotein became resistant to protease digestion when the glycoprotein was inserted into the vesicle bilayer. When the covalently attached fatty acid of the glycoprotein was labeled in vivo with [3H]lpalmitate, it was demonstrated that the fatty acid was attached to the hydrophobic glycoprotein tail fragment in vesicle and virion membranes.

The exchangeability of human erythrocyte membrane cholesterol.

A new method has been used to determine what fraction of human erythrocyte cholesterol is available for exchange with plasma unesterified cholesterol. Erythrocytes labeled with 3H-cholesterol by this exchange process were incubated with sonicated phosphatidylcholine vesicles, giving rise to a net movement of cholesterol out of the cells. The specific activity of cholesterol taken up by the vesicles depended on the length of time of incubation. Initially the specific activity in the vesicles was greater than that in the cells, but after approximately 10% of cell cholesterol had been removed, the specific activity of subsequently removed cholesterol was equal to that of the remaining erythrocyte cholesterol. We conclude from these data that a) all of the cholesterol in the erythrocyte is exchangeable with plasma, and b) approximately 10% of erythrocyte cholesterol is in a more rapidly exchangeable pool than the remainder.

13C NMR studies on [4- 13C]cholesterol incorporated in sonicated phosphatidylcholine vesicles

1. 1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4- 13C]cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. 2. From the Dy 3+-induced shift of the C 4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. 4. At cholesterol concentrations below 30 mol% the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.

Selective extraction of membrane-bound proteins by phospholipid vesicles.

Extraction of membrane proteins from erythrocytes into sonicated phosphatidylcholine vesicles is described. In a process involving phospholipid and neutral lipid exchange, cell membrane proteins associate with the vesicles and can be separated from the cells by centrifugation. The protein transfer appears to be reversible; phospholipid vesicles mediate the delivery of small amounts of previously extracted protein into cell membranes. Prior to extraction, all but one of the proteins are accessible to lactoperoxidase iodination, and lipid analysis indicates that primarily the outer monolayer of the cell is involved in phospholipid exchange. Among the extracted proteins is acetylcholinesterase which is removed much more efficiently by this procedure than by concentrated salt solutions. The most abundant proteins of the erythrocyte membrane are not represented in the vesicle extract.