Effect of bilayer membrane curvature on activity of phosphatidylcholine exchange protein

Effect of bilayer membrane curvature of substrate phosphatidylcholine and inhibitor phosphatidylserine on the activity of phosphatidylcholine exchange protein has been studied by measuring transfer of spin-labeled phosphatidylcholine between vesicles, vesicles and liposomes, and between liposomes. The transfer rate between vesicles was more than 100 times larger than that between vesicles and liposomes. The transfer rate between liposomes was still smaller than that between vesicles and liposomes and nearly the same as that in the absence of exchange protein. The markedly enhanced exchange with vesicles was ascribed to the asymmetric packing of phospholipid molecules in the outer layer of the highly curved bilayer membrane. The inhibitory effect of phosphatidylserine was also greatly dependent on the membrane curvature. The vesicles with diameter of 17 nm showed more than 20 times larger inhibitory activity than those with diameter of 22 nm. The inhibitory effect of liposomes was very small. The size dependence was ascribed to stronger binding of the exchange protein to membranes with higher curvatures. The protein-mediated transfer from vesicles to spiculated erythrocyte ghosts was about four times faster than that to cup-shaped ghosts. This was ascribed to enhanced transfer to the highly curved spiculated membrane sites rather than greater mobility of phosphatidylcholine in the spiculated ghost membrane.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Phosphatidylcholine Asymmetry in Electroplax from the Electric Eel: Use of a Phosphatidylcholine Exchange Protein

Phosphatidylcholine asymmetry in the inner and outer leaflets of the plasma membrane bilayer of the innervated and noninnervated surfaces of the electroplax cell was determined, using a Phosphatidylcholine exchange protein. The exchange protein from bovine liver catalyzed the exchange of Phosphatidylcholine from small unilamellar vesicles to the outer monolayer of the plasma membrane bilayer. The exchange protein did not penetrate to the inner monolayer of the plasma membrane, did not modify the permeability of the electroplax, and did not alter the phospholipid or cholesterol content of the electroplax. In the innervated plasma membrane, 42% of the Phosphatidylcholine is in the outer leaflet, 33% is in the inner leaflet, and 25% is inaccessible to the exchange protein. Corresponding values for the noninnervated plasma membrane are 56, 26, and 18%, respectively. These results are similar to Phosphatidylcholine asymmetry in other biological membranes. This unique cell can be used as a model to test the effects on phospholipid asymmetry of compounds that act on the membrane.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.     

The sphingomyelin pools in the outer and inner layer of the human erythrocyte membrane are composed of different molecular species

Analyses of the fatty acid composition of the outer and inner pools of sphingomyelin in the human erythrocyte membrane revealed significant differences in molecular species composition of these two pools. The sphingomyelin in the inner monolayer, representing 15–20% of the total sphingomyelin content of this membrane, is characterized by a relatively high content (73%) of fatty acids, which have less than 20 carbon atoms, whereas these account for only 31% of the total fatty acids in the sphingomyelin in the outer leaflet. On the other hand, the ratio saturated/unsaturated fatty acids in the two pools is similar. Significant differences are also observed for the fatty acid composition of the sphingomyelin in human serum when compared to that in the outer monolayer of the corresponding red cell. These results are interpreted to indicate an (almost) complete absence of transbilayer movements of sphingomyelin molecules in the human erythrocyte membrane, whereas an exchange of this phospholipid between the red cell membrane and serum is either virtually absent, or affects only a minor fraction of the sphingomyelin in the outer membrane layer.     

Inhibition of the binding of cytochrome b5 to phosphatidylcholine vesicles by cholesterol

The binding of cytochrome b₅ to single-walled liposomes of egg phosphatidylcholine was inhibited by the presence of cholesterol in the lipid bilayer under conditions where a limited amount of liposomes was incubated with the cytochrome. Since similar conditions seem to apply for the binding of cytochrome b₅ to erythrocyte ghosts, this observation supports the conclusion of Enomoto and Sato (Enomoto, K. and Sato, R. (1977) Biochim. Biophys. Acta 466, 136–147) that the localization of cholesterol on the outer surface of the ghost membrane prevents the binding of cytochrome b₅ to this surface. The finding reported by Roseman et al. (Roseman, M.A., Holloway, P.W. and Calabro, M.A. (1978) Biochim. Biophys. Acta 507, 552–556) that cholesterol did not prevent the cytochrome binding to phosphatidylcholine liposomes in the presence of a large excess of liposomes could be confirmed in the present study, but this does not contradict the abovementioned conclusion.     

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles

We have characterized the asymmetric effect of Ca²⁺ on passive K⁺ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca²⁺, but not Mg²⁺, can induce an increase in K⁺ uptake in inside out vesicles. The half-maximal concentration of Ca²⁺ required to induce the K⁺ uptake is 0.2 mM, and the permeability increase is not specific for K⁺. Thus, the Ca²⁺-induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca²⁺ and is specific for K⁺. Removal of spectrin had no effect on the vesicle permeability increase due to Ca²⁺. Studies with $\text{N-}\text{ethylmaleimide}$ show that the vesicle channel opening is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca²⁺-induced vesicle pathway is distinct from the normal channel for passive K⁺ leak in the absence of Ca²⁺. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca²⁺-stimulated channel opening.     

Effect of Soman and Sarin on Phosphatidylcholine Asymmetry in the Electroplax from the Electric Eel

Some effects of organophosphorus anticholinesterase compounds that are unrelated to cholinesterase inhibition and that are sometimes long lasting may be due to alterations at the cellular membrane level. Phosphatidylcholine exchange protein was used to assess the effects of sarin and soman on phosphatidylcholine asymmetry in the inner and outer leaflets of the plasma membrane bilayer of the electroplax. Exposure of electroplax (30 min in vitro) to soman (10(-4), 10(-6) M) or sarin (10(-4), 10(-6), 5 x 10(-9) M) increased the percentage of phosphatidylcholine in the outer monolayer of the innervated plasma membrane bilayer and decreased the percentage in the inner monolayer. These changes by sarin were observed at concentrations that produced 100% cholinesterase inhibition (10(-4), 10(-6) M) and at a concentration (5 x 10(-9) M) where no inhibition occurred, suggesting that these effects are not directly due to cholinesterase inhibition. A 1-week exposure of live eels to soman (10(-8) M) in vivo caused an increase in phosphatidylcholine labeling in the outer monolayer of the innervated and noninnervated surfaces of the electroplax. Two weeks after stopping exposure to soman, increased labeling was still observed, suggesting that this may be a long-term effect. Because the organophosphates did not increase the permeability of the electroplax, all of these changes in labeling appear to be due to a redistribution of phosphatidylcholine from the inner to the outer monolayer of the plasma membrane bilayer.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

Permeability of human erythrocyte membrane vesicles to alkali cations

The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using ⁸⁶Rb as a K analog, we have measured the rate constant of ⁸⁶Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.     

Detection of phosphatidylcholine oxidation products in rat heart using quadrupole time-of-flight mass spectrometry

An improved technique for the analysis of phosphatidylcholine (PC) and lyso-phosphatidylcholine (lyso-PC) oxidation products was developed using quadrupole time of flight (Q-TOF) mass spectrometry with electrospray ionization. We separated these products using an HPLC C(8) column with a gradient of methanol and 10mM aqueous ammonium acetate. Monohydroxides, oxo derivatives, and trihydroxides of palmitoyl-linoleoyl (C16:0/C18:2) PC, stearoyl-linoleoyl (C18:0/C18:2) PC, and oleoyl-linoleoyl (C18:1/C18:2) PC were detected mainly as MH(+) and [M+Na](+) ions in the heart of the intact rat. Using standard synthetic PC-OH (C16:0/C18:2-OH), the lipid extract component was identified as (C16:0/C18:2-OH) PC based on the product ions of ESI-MS-MS and, the PC-OH concentration was quantitated. Four oxidatively modified 1-lyso-phosphatidylcholines (lyso-PCs) were also detected. This is the first report showing the presence of monohydroxides, oxo derivatives, and trihydroxides of (C16:0/C18:2)PC, (C18:0/C18:2)PC, and (C18:1/C18:2) PC in the rat heart.     

The membrane lipid phosphatidylcholine is an unexpected source of triacylglycerol in the liver

The increased prevalence of obesity and diabetes in human populations can induce the deposition of fat (triacylglycerol) in the liver (steatosis). The current view is that most hepatic triacylglycerols are derived from fatty acids released from adipose tissue. In this study, we show that phosphatidylcholine (PC), an important structural component of cell membranes and plasma lipoproteins, can be a precursor of ~65% of the triacylglycerols in liver. Mice were injected with [(3)H]PC-labeled high density lipoproteins (HDLs). Hepatic uptake of HDL-PC was ~10 μmol/day, similar to the rate of hepatic de novo PC synthesis. Consistent with this finding, measurement of the specific radioactivity of PC in plasma and liver indicated that 50% of hepatic PC is derived from the circulation. Moreover, one-third of HDL-derived PC was converted into triacylglycerols. Importantly, ~65% of the total hepatic pool of triacylglycerol appears to be derived from hepatic PC, half of which is derived from HDL. Thus, lipoprotein-associated PC should be considered a quantitatively significant source of triacylglycerol for the etiology of hepatic steatosis.     

A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts

A commercially available enzymatic assay (Boehringer Monotest) was modified to allow a rapid and sensitive determination of cholesterol in membrane lipid extracts. This was achieved by adding 0.5% Triton X-100 to the reagent solution. The detergent did not interfere with the assay. The relationship between the amount of cholesterol per assay and the absorbance at 500 nm was linear up to 100 μg. The recovery in the assay was better than 95%. The assay was applied to the determination of cholesterol in erythrocyte membrane lipid extracts.     

Comparison of cholesterol-phospholipid interaction in bilayers of liposomes and the red cell membrane

The energetics of interactions of cholesterol with phospholipid in simple liposome bilayers were compared with those in the bilayer of the human erythrocyte membrane, by measuring cholesterol distribution between erythrocytes and liposomes prepared from their whole phospholipid extract. With liposomes of a range of initial cholesterol contents, the equilibrium value for $\text{r}$, the ratio of cholesterol/phospholipid in the liposomes to that in the cells, is in the range 1.1–1.2. The closeness of this value to 1.0 indicates that overall cholesterol-phospholipid interaction in the cell membrane is similar to that in liposomes. However, while the deviation from 1.0 is small, and could arise from average cholesterol-phospholipid interactions in the membrane being only 0.06 to 0.1 kcal · mol^?1 weaker than in liposomes, it could also result from 10 to 20% of the cell membrane phospholipid being unavailable to mix with cholesterol.     

Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa (PACT) in rat erythrocytes and black lipid membranes

A cytotoxic protein, isolated from Pseudomonas aeruginosa (PACT), was tested on red blood cells of rats and on black lipid membranes for changes of membrane permeability. In rat erythrocytes PACT induces lysis indicative of the formation of a leak permeable to monovalent ions. The dose response curve for the PACT-induced hemolysis demonstrates that the rate of lysis as well as the fraction of lytic cells increases with increasing toxin concentration. Furthermore, the leak pathway discriminates hydrophilic non-electrolytes according to their molecular weight. The findings indicate formation by PACT of a pore with an apparent radius of about 1.2 nm. In pure lipid membranes PACT forms hydrophilic pathways with moderate selectivity for small cations over small anions. The presence of cholesterol is a prerequisite for the occurrence of these PACT-induced permeability changes.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture

We studied the effect of fetal calf serum and serum protein fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine β-globulins reduced this transfer, although to a lesser extent than whole serum. α-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A γ-globulin fraction failed to exhibit any effect on the uptake of [¹⁴C]phosphatidylcholine by the cells.All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles preincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine to parenchymal cells.These observations were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of the liposomal surface membrane as a result of the binding of certain serum proteins. On the other