The affinity of cholesterol for phosphatidylcholine and sphingomyelin.

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for N-palmitoyl sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.     

Asymmetric binding of cytochrome b5 to the membrane of human erythrocyte ghosts

The intact, amphipatic form of cytochrome $\text{b}{}_5$ could bind to unsealed ghosts, but not to resealed ghosts, suggesting that the cytochrome could bind only to the inner (cytoplasmic) surface of the ghost membrane. This was further confirmed by the finding that the cytochrome could bind to closed, inside-out vesicles prepared from the ghosts. This asymmetric binding was not due to the exclusive localization of sialic acid and sugar chains on the outer surface of the ghosts membrane, because the cytochrome could not bind to ghosts even after enzymatic removal of these components. Although liposomes consisting of phosphatidylcholine or both phosphatidylcholine and sphingomyelin could effectively bind the cytochrome, this binding capacity was progressively decreased as increasing amount of cholesterol was included in the composition of phosphatidylcholine liposomes. Removal of cholesterol from resealed ghosts by incubation with egg phosphatidylcholine liposomes resulted in the binding of cytochrome $\text{b}{}_5$ to the outer surface of the treated ghosts. The possibility is discussed that the asymmetric binding is due to preferential localization of cholesterol in the outer leaflet of the lipid bilayer that constitutes the ghost membrane.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

Phase transitions in phospholipid vesicles Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol

We have studied the solid to liquid-crystalline phase transition of sonicated vesicles of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine. The transition was studied by both fluorescence polarization of perylene embedded in the vesicles, and by the efflux rate of trapped ²²Na⁺.Fluorescence polarization generally decreases with temperature, showing an inflection in the region 32–42°C with a mid-point of approximately 37.5 °C. On the other hand, the perylene fluorescence intensity increases abruptly in this region. To explain this result, we have proposed that, for $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ where $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ is the transition temperature, perylene is excluded from the hydrocarbon interior of the membranes, whereas, $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ this probe may be accommodated in the membrane interior to a large extent.The self-diffusion rates of ²²Na⁺ through dipalmitoylphosphatidylglycerol vesicles exhibit a complex dependence on temperature. There is an initial large increase in diffusion rates (approximately 100-fold) between 30 and 38 °C, followed by a decrease (approximately 4-fold) between 38 and 48 °C. A monotonic increase is then observed at temperatures higher than 48 °C. The local maximum of ²²Na⁺ self-diffusion rates at approximately 38 °C coincides with the mid-point of phase transition as detected by changes in fluorescence polarization of perylene with the same vesicles. Vesicles composed of dipalmitoylphosphatidylcholine show the same general behavior in terms of ²²Na⁺ self-diffusion rates at different temperatures, except that the local maximum occurs at approximately 42 °C.The temperature dependence of the permeability and the appearance of a local maximum at the phase transition region could be explained in terms of a domain structure within the plane of the membranes. This explanation is based on the possibility that boundary regions between liquid and solid domains would exhibit relatively high permeability to ²²Na⁺.Mixed vesicles composed of equimolar amounts of dipalmitoyl phospholipids and cholesterol show no abrupt changes in the temperature dependence of either perylene fluorescence polarization or ²²Na⁺ diffusion rate measurements. This is taken to indicate the absence of agross phase transition in the presence of cholesterol.     

Role of the plasma membrane in the mechanism of cholesterol efflux from cells

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [³H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu₅AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for plasma membrane vesicles of Fu₅AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu₅AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu₅AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values for cholesterol efflux from these cell lines.     

Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a $\text{p}\text{K}$ of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.     

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10–12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with $\text{S-}\text{adenosyl}\text{-[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the formation of $\text{phosphatidyl}\text{-N-}\text{monomethylethanolamine}$, $\text{phosphatidyl}\text{-N,N-}\text{dimethylethanolamine}$ and phosphatidylcholine from endogenous phosphatidylethanolamine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4–5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to $\text{phosphatidyl}\text{-N-}\text{monomethylethanolamine}$ or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na⁺-dependent uptake of either d-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.     

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, $\text{η}$, of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, $\text{η}$ was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells $\text{η}$ values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types $\text{η}$ (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.     

Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexose transfer

(1) The Michaelis-Menten parameters for hexose transfer in erythroctes, erythrocyte ghosts and inside-out vesicles at 20°C were determined using the light scattering method of Sen and Widdas ((1962) J. Physiol. 160, 392–403). (2) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit of d-glucose in cells and ghosts is $\text{3.6 ± 0.5}\text{mM}$. (3) Dilution of cellular solute (up to × 90 dilution) by lysing and resealing cells in varying volumes of lysate is without effect on the $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net d-glucose exit. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for net exit, however, falls from $\text{32.4 ± 3.7}\text{mM}$ in intact cells to $\text{12.9 ± 2.3}\text{mM}$ in ghosts. This effect is reversible. (4) Infinite-cis net d-glucose uptake measurements in cells and ghosts reveal the presence of a low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, high affinity internal site of $\text{5.9 ± 0.8}\text{mM}$. The $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net glucose entry increases from $\text{23.2 ± 3.7}\text{mmol/l per min}$ in intact cells to $\text{55.4 ± 6.3}\text{mmol/l per min}$ in ghosts. (5) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cisd-glucose exit in inside-out vesicles is $\text{6.8 ± 2.7}\text{mM}$. The kinetics of zero-transd-glucose exit from inside-out vesicles are changed markedly when cellular solute (obtained by lysis of intact cells) is applied to either surface of inside-out vesicles. When solute is present externally, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit are decreased by up to 10-fold. When solute is present at the interior of inside-out vesicles, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit is reduced; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for exit is unaffected. In the nominal absence of cell solute, transfer is symmetric in inside-out vesicles. The orientation of transporter in the bilayer is unaffected by the vesiculation procedure. (6) External application of cellular solute to ghosts reduces $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for d-glucose exit but is without effect on the external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit. (7) The inhibitory potency of cell lysate on hexose transfer is lost following dialysis indicating that the factors responsible for transfer modulation are low molecular weight species. (8) We consider the hexose transfer in human erythrocytes is intrinsically symmetric and that asymmetry of transfer is conferred by interaction of the system with low molecular weight cytosolic factors.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Transbilayer movement of cholesterol in phospholipid vesicles under equilibrium and non-equilibrium conditions

1. The exchange of [3H] cholesterol between phospholipid: cholesterol vesicles and an excess of red cell ghosts is examined. 2. Using a number of different phophatidylcholines, only the cholesterol thought to be associated with the outer half of the bilayer (about 70 percent) is available for exchange, suggesting that at least at equilibrium the transbilayer movement of cholesterol or "flip-flop", occurs very slowly, if it occurs at all. 3. The rate of exchange of cholesterol between the vesicles and the ghosts is dependent on the nature of the fatty acid chain of the phospholipids, being a function of both the fatty acid chain length and the degree of unsaturation. 4. Under non-equilibrium conditions, when cholesterol is being both exchanged and depleted from the lipid vesicles to red cell ghosts, the previously non-exchangeable vesicle cholesterol becomes available for exchange, suggesting that under these conditions "flip-flop" can occur.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

Preparation and structural studies of cholesterol bilayers

Bilayers consisting, in their hydrophobic core, entirely of cholesterol can be constructed if a hydrophilic molecular anchor is supplied. $\text{O-}\text{Methoxyethoxyethoxyethylcholesterol}$ and cholesterol sulfate form multilayered liposomes in water. With equimolar cholesterol added, cholesterol sulfate, cholesterolphosphocholine, and $\text{O-}\text{methoxyethoxyethoxyethylcholesterol}$ form small unilamellar liposomes on prolonged sonication. The dimensions of cholesterol-cholesterolphosphocholine vesicles are comparable to those of phospholipid vesicles. ¹³C-NMR spectra suggest that the centers of the bilayers are liquid. The permeability of the cholesterol-cholesterolphosphocholine bilayer against glycerol is lower than that of dipalmitoylphosphatidylcholine-cholesterol bilayer; the activation energy of permeation is two times larger, an indication of a higher degree of structural organization in the ‘hydrogen belts’ of the cholesterol-cholesterolphosphocholine bilayer.     

Microvillus membrane vesicles from pig small intestine Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18:50:29:2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17,17,6,5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either $\text{N-}\text{acetylglucosamine}$ or $\text{N-}\text{acetylgalactosamine}$) in the molar ratio of 1:2:1:1.     

Interaction of DDT (1,1,1-trichloro-2,2-BIS(p-chlorophenyl)-ethane with liposomal phospholipids

The influence of $\text{1,1,1-}\text{trichloro}\text{-2,2-}\text{bis}\text{(p-}\text{chlorophenyl}\text{)}\text{ethane}$ (DDT) and several other pesticides on the physical state of membrane phospholipids was investigated using model lipids. The thermal dependence of fluorescence intensity of the probe parinaric acid in dipalmitoylphosphatidylcholine liposomes and lipid vesicles of mixed composition were recorded. DDT was incorporated into the liposomal bilayer. The insecticide lowered the phase transition temperature and broadened the temperature range of the transition. The effects were concentration-dependent.The results may be interpreted as a sort of blurred and facilitated phase transition of bilayer lipids caused by intercalation of DDT between fatty acyl chains of membrane phospholipids.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Active calcium transport in red cell ghosts resealed in dextran solutions

1.Human erythrocytes when lysed and resealed to Ca in the presence of dextran can be readily separated from the suspending medium by low-speed centrifugation. 2. Ghosts trapped Ca and EGTA at the same ratio as present in the haemolytic medium and remained tight to Ca after washing and subsequent incubation for up to 90 min at 37°C. 3. Ca extrusion could be promoted by substrates other than ATP only from ghosts that had been loaded with low free Ca concentrations (1–22 μM). The order of activation by the various substrates employed was $\text{ATP}\text{>}\text{adenine}\text{+}\text{inosine}\text{>}\text{inosine}$. 4. The kinetics of extrusion depended markedly on internal free Ca. The system showed a high affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{3 μ}\text{M}$; $\text{V = 0.34 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at low concentrations (1–22 μM) and a low affinity state ($\text{K}{}_{\mathrm{<mtext>Ca</mtext>}}\text{about}\text{250 μ}\text{M}$; $\text{V = 0.17 μ}\text{mol Ca}\text{/}\text{ml ghosts per min}$) at high concentrations (0.2–4.0 mM). 5. Both at low and at high free Ca, La-sensitive ATP hydrolysis was closely correlated with La-dependent Ca efflux, in keeping with an stoichiometry of 1. 6. The rate of extrusion was maximal in the presence of 160 mM KCl and decreased to various extents when K was fully replaced by different cations, following the order $\text{K}\text{>}\text{Na = choline}\text{>}\text{Mg}$. 7. The efflux rate of high-K ghosts, resealed to alkaline cations, was stimulated by external Na, whilst Mg and choline were practically without effect. 8. The results indicate that human red cells possess a powerful Ca extrusion mechanism, the activity of which can be modulated by alkaline cations.     

Changes in lipid composition of cortical synaptosomes from different age groups of mice

Lipid composition of cortical synaptosomes differed with age in C57BL/6NNIA mice. Significant age differences were observed for cholesterol and the ratio of cholesterol to total phospholipid phosphorus content. The phospholipid to protein ratio of individual phospholipids also increased with age with diacyl-$\text{sn}$-glycero-3-phosphocholine (PC) increasing the most. Acyl group composition of individual phospholipids, however, showed little age difference. The double bond index for PC decreased significantly with age. Changes in membrane composition may help explain differences in the effects of ethanol on the physical and biochemical properties of membranes from different age groups that have been reported previously.