Reaction of ozone with phospholipid vesicles and human erythrocyte ghosts

Phospholipid vesicles (unilamellar) and liposomes (multilamellar) made from egg phosphatidylcholine reacted similarly with ozone, producing hydrogen peroxide and malonaldehyde. On the basis of amount of ozone reacted, there was a 20% yield of hydrogen peroxide and 2.4% yield of malonaldehyde. The reactivity of the egg phosphatidylcholine membranes was a function of exposed membrane surface area. Large amounts of ozone caused no change in erythrocyte ghost phospholipid, fatty acid, or cholesterol composition. Thiobarbituric acid-positive material and conjugated dienes were present in very small quantities, suggesting some lipid oxidation which was below the limits of chromatographic detection. Ozone inhibited glyceraldehyde 3-phosphate dehydrogenase more than (Na⁺ + K⁺) adenosine triphosphate in exposed unsealed erythrocyte ghosts. The (Na⁺ + K⁺) adenosine triphosphatase activity sensitive to ozone was the ouabain-insensitive activity. Acetylcholinesterase activity was not significantly inhibited.     

The mixture of aldehydes and hydrogen peroxide produced in the ozonation of dioleoyl phosphatidylcholine causes hemolysis of human red blood cells

Dioleoyl phosphatidylcholine (PC) liposomes were ozonized and the ozonized liposomes were tested for their lytic potency on human red blood cells (RBC). Ozonation of PC liposomes generated approximately 1 mole equivalent of hydrogen peroxide (H2O2) and 2 mole equivalents of aldehydes, based on the moles of ozone consumed. The time necessary for 50% hemolysis induced by ozonized liposomes (a convenient measure of hemolytic activity) was found to depend on the extent of ozonation of the PC liposomes, indicating the formation and accumulation of hemolytic agents during ozonation. Hemolysis was also observed when RBC were incubated with nonanal, the expected product of the ozonation of oleic acid, the principle unsaturated fatty acid in the liposomes. Hydrogen peroxide, another product of PC ozonation, did not induce hemolysis; however, a combination of H2O2 and nonanal was significantly more hemolytic than nonanal alone. A ratio of 1:2 H2O2/nonanal (the ratio observed in the ozonized liposomes) provided hemolytic activity comparable to that observed with ozonized dioleoyl PC. Among different antioxidants tested, ascorbate, catalase, and glutathione peroxidase partially inhibited hemolysis induced by ozonized liposomes and by H2O2/nonanal mixtures, but they were not protective against the nonanal-induced hemolysis. Identification of H2O2 and aldehydes as cytotoxic chemical species generated from the ozonation of unsaturated fatty acids may have an important bearing on the in vivo toxicity of ozone on the lung as well as on extrapulmonary tissues.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

Disintegration of phosphatidylcholine liposomes in plasma as a result of interaction with high-density lipoproteins.

1. During in vitro incubation of liposomes or unilamellar vesicles prepared from egg-yolk or rat-liver phosphatidylcholine with human, monkey or rat plasma the phospholipid becomes associated with a high molecular weight protein-containing component. 2. The phosphatidylcholine . protein complex thus formed co-chromatographs with high-density lipoprotein on Ultrogel AcA34 and has the same immunoelectrophoretic properties as this lipoprotein. 3. Release of the phosphatidylcholine from liposomes was also observed when liposomes were incubated with pure monkey high-density lipoproteins. Under those conditions some transfer of protein from the lipoprotein to the liposomes was observed as well. 4. The observed release of phospholipid from the liposomes is a one-way process, as the specific radioactivity of liposome-associated phosphatidylcholine remained constant during incubation with plasma. 5. It is concluded that either the lipoprotein particle takes up additional phospholipid or that a new complex is formed from protein constituents of the lipoprotein and the liposomal phosphatidylcholine. 6. Massive release of entrapped 125I-labeled albumin from the liposome during incubation with plasma suggests that the observed release of phosphatidylcholine from the liposomes has a highly destructive influence on the liposomal structure. 7. Our results are discussed with special reference to the use of liposomes as intravenous carriers of drugs and enzymes.     

Liposomal Drug with Carnosine and Lipoic Acid: Preparation,Antioxidant and Antiplatelet Properties

The conditions for producing phosphatidylcholine liposomes containing lipoic acid and carnosine together were determined. The obtained liposomes are 180–250-nm spherical particles with an efficiency of lipoic acid inclusion of 50–70% (for carnosine, 17–33%). Based on the model of the oxidation of phosphatidylcholine by hydrogen peroxide, an antioxidant effect of carnosine, lipoic acid or lipoic acid with carnosine together was demonstrated; it consisted in inhibition of lipid peroxidation process, which was manifested in a decrease in the formation of lipid peroxidation products that react with thiobarbituric acid. It was established that lipoic acid (5 mM) and carnosine (0.1–10 mM) in liposomes exhibit an antioxidant effect. At the same time, it was demonstrated that the content of the appropriate lipid peroxidation products in liposomes with antioxidants (lipoic acid + carnosine) was 15 times lower than in control liposomes (without antioxidants). The effect of the obtained liposomal drugs on the platelet aggregation induced by arachidonic acid was evaluated. It was found that the liposomal drug containing lipoic acid (1.5 mM) and carnosine (2.1 mM) inhibited platelet aggregation by 50–55% relative to the control (platelets and arachidonic acid), while liposomes without antioxidants and water-soluble forms of carnosine and lipoic acid had almost no effect on platelet aggregation caused by arachidonic acid.

     

Permeability Changes by Peroxidation of Unsaturated Liposomes with Ascorbic Acid/Fe2+

Abstract

Liposomes composed of phosphatidylcholine having a polyunsaturated fatty acid side chain were peroxidized in ascorbic acid/Fe²⁺ solution. Lipid peroxidation and the change in membrane permeability were monitored by the formation of thiobarbituric acid reactive substance (TBARS) and the release of entrapped fluorescein isothiocyanate-labeled superoxide dismutase (FITC-SOD), respectively. Peroxidation of liposomes composed of dipalmitoylphosphatidylcholine and 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) having 4 double bonds on one fatty acid side chain showed high TBARS value and caused the release of FITC-SOD. This release started when TBARS reached a definite value. But liposomes composed of phosphatidylcholine having 1 or 2 double bond(s) on one fatty acid side chain caused little increase in lipid peroxidation and FITC-SOD release. During the peroxidation of PAPC-liposomes, the breakdown of PAPC and formation of lysophosphatidylcholine (or like substance) were detected by HPLC analysis. Increase in the release of FITC-SOD thus appears to be due to the breakdown of the fatty acid side chain of phospholipids of liposomes. Liposomes composed of phosphatidylcholine having a polyunsaturated fatty acid side chain may be expected to be sensitive to peroxidation signals.     

Liposomes of dipalmitoylphosphatidylcholine associate with natural surfactant

Unilamellar liposomes of an average diameter of 0.05 micron formed by sonication of dipalmitoylphosphatidylcholine associate in vitro with the large aggregate forms of natural surfactant. The liposomal-surfactant aggregates are stable and previously associated liposomes are not released from the aggregates by the addition of more liposomes. Radiolabeled liposomes, surfactant, and preformed liposomal-surfactant aggregates were injected at a dose of 8-10 mg lipid (about 2-times the endogenous surfactant pool size) into the airways of 3-day-old rabbits. Following airway injection, labeled phosphatidylcholine from the liposomal-surfactant aggregates were recovered in approximately equal amounts by alveolar wash and in the residual lung tissue fractions. This recovery pattern and the clearance kinetics were equivalent for 48 h after airway injection to those measured with radiolabeled surfactant alone. In contrast, following the injection of liposomes alone, labeled phosphatidylcholine from the liposomes was recovered primarily by alveolar wash at 3 and 24 h. The overall clearance of the liposomal-derived phosphatidylcholine from the lung was more rapid than was the clearance of the phosphatidylcholine from the surfactant or liposome-surfactant complexes. Liposomes can interact with surfactant in vitro, and the liposomes associated with the surfactant aggregate have a metabolic fate in vivo similar to surfactant and different from liposomes alone.     

Sequence specificity of ozone-degradation of bases in supercoiled plasmid DNA

It was found that ozone reacted preferentially with thymine and guanine residues located in the specific region in pBR322 DNA. The sequence analysis of the region including the cleavage site produced by ozonization of ccDNA showed that ozone-modification proceeded in the single stranded region formed by cruciform-formation in supercoiled DNA.     

Transfer of cholesterol between liposomal membranes.

The transfer of cholesterol between liposomal membranes was examined. On incubation of liposomes compsoed of egg yolk phosphatidylcholine, phosphatidic acid and cholesterol (molar percentage, 65.8 : 1.3 : 32.9 or 65.5 : 6.3 : 31.2), almost complete equilibration of the cholesterol pools was achieved within 6 to 8 h at 37 degrees C. The rate of transfer of cholesterol from the liposomes, in which cholesterol was introduced by 'the exchange reaction', was not significantly different from that from liposomes prepared in the presence of cholesterol, in which the cholesterol was distributed homogenously. These findings indicate that half life for 'flip-flop' of cholesterol molecules in egg yolk phosphatidylcholine liposomes is less than 6 h at 37 degrees C. The transfer of cholesterol between liposomes was strongly dependent on temperature and was affected by the fatty acid composition of the phospholipid, suggesting that the 'fluidity' of the membranes strongly influences the transfer rate. A preferential distribution of cholesterol molecules was observed in heterogeneous liposomes with different classes of phospholipids. The 'affinity order' of cholesterol for phospholipid deduced from the present experiments is as follows: beef brain sphingomyelin greater than dipalmitoylglycerophosphocholine = dimyristoylglycerophosphocholine greater than egg yolk phosphatidylcholine.     

Characteristics of Sendai virus receptors in a model membrane

The adsorption of Sendai virus to liposomes of different compositions was studied. Liposomes prepared with only phosphatidylcholine and cholesterol, and liposomes prepared with phosphatidylcholine and cholesterol plus phosphatidic acid or phosphatidyl serine did not adsorb virus. Phosphatidyleholine-cholesterol liposomes containing also stearyl amine or ganglioside did, however, adsorb virus. The ability of the adsorbing liposomes to compete with erythrocytes for virus was measured by hemagglutination inhibition. Liposomes containing ganglioside, but not those containing stearyl amine, inhibited hemagglutination. When the molar ratio of ganglioside N-acetyl neuraminic acid to phosphatidylcholine was less than 0.02, ganglioside liposomes did not inhibit hemagglutination. As the ratio increased from 0.02 to 0.05, the liposomes caused increasing amounts of hemagglutination inhibition, but with further increases in the ratio the hemagglutination inhibition remained constant. It is concluded that gangliosides can serve as Sendai receptors and that a multiplicity of receptors is needed for virus binding.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

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.     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

Kinetic analysis of the interaction of the phosphatidylcholine exchange protein with unilamellar vesicels and multilamellar liposomes.

The mode of action of the phosphatidylcholine exchange protein from bovine liver has been studied by using unilamellar vesicles and multilamellar liposomes both of which membranes contain phosphatidylcholine and phosphatidic acid. The protein-mediated exchange of phosphatidylcholine between vesicles and liposomes fit the kinetic model presented in a previous study [V.D. Besselaar et al. (1975) Biochemistry, 1j, 1852]. Kinetic analysis of the rates of exchange indicate that the apparent dissociation constant of the exchange protein-vesicle complex decreases with an increasing phosphatidic acid content of the vesicles. Both vesicles and liposomes of 10 mol% phosphatidic acid show the same dissociation constant; on the other hand, both the formation and the disruption of the protein-membrane complex was 50--100-times higher for the vesicles than for the liposomes. This implies that the exchange protein can discriminate between vesicles and liposomes. Equilibrium gel chromatography of a column of Bio Gel A-5m confirmed that the exchange protein binds more strongly to vesicles of an increased phosphatidic acid content. The protein-mediated exchange of phosphatidylcholine in the vesicle-liposome system demonstrates a pH optimum at 4.0 to 5.5. The kinetic analysis at pH 5.0 as compared to pH 7.4 indicates that the enhanced exchange at pH 5.0 can solely be accounted for by altered interaction of the exchange protein with the liposomes.     

Steady-state fluorescence anisotropy and multifrequency phase fluorometry on oxidized phosphatidylcholine vesicles

Multilamellar liposomes, from mixtures of unoxidized (control) and singlet oxygen oxidized phosphatidylcholine, were studied by steady-state fluorescence anisotropy and multifrequency phase fluorometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Lifetime fluorescence decay of the DPH-labeled liposomes was analyzed either by a model of discrete exponential components and a model that assumes a continuous distribution of lifetime values. Increasing the oxidized phosphatidylcholine content in the liposomes, an increase of the membrane interior polarity and a decrease of membrane fluidity occurs which can be related to the hydroperoxide-lipids and double bonds conjugation, respectively.     

Hemolysis of phosphatidylcholine-containing erythrocytes by serratamic acid from Serratia marcescens.

1. The hemolysis by serratamic acid, "N-(D-3-hydroxydecanoyl)-L-serine and N-(D-3-hydroxydodecanoyl)-L-serine", was investigated with human and animal erythrocytes using serratamic acid-containing liposomes. 2. The hemolytic activity was found to depend on the incubation temperature and the concentration of the liposomes. 3. The concentration of serratamic acid for 50% hemolysis was 0.17 mM at 37 degrees C for 0.2% human erythrocyte suspension in the liposomes which composed of phosphatidylserine, cholesteryl nervonate and serratamic acid (1:0.50:0.37 by mol). 4. The hemolysis was shown specifically in human, horse and rabbit erythrocytes containing phosphatidylcholine, but not in sheep or bovine erythrocytes lacking phosphatidylcholine. 5. The hemolytic activity was strongly inhibited by the exogenous addition of phosphatidylcholine. It was suggested that the hemolysis by serratamic acid-containing liposomes was specific for phosphatidylcholine-containing erythrocyte membranes.     

Fusion of Sendai virions or reconstituted Sendai virus envelopes with liposomes or erythrocyte membranes lacking virus receptors

Incubation of intact Sendai virions or reconstituted Sendai virus envelopes with phosphatidylcholine/cholesterol liposomes at 37 degrees C results in virus-liposome fusion. Neither the liposome nor the virus content was released from the fusion product, indicating a nonleaky fusion process. Only liposomes possessing virus receptors, namely sialoglycolipids or sialoglycoproteins, became leaky upon interaction with Sendai virions. Fusion between the virus envelopes and phosphatidylcholine/cholesterol liposomes was absolutely dependent upon the presence of intact and active hemagglutinin/neuraminidase and fusion viral envelope glycoproteins. Fusion between Sendai virus envelopes and phosphatidylcholine/cholesterol liposomes lacking virus receptors was evident from the following results. Anti-Sendai virus antibody precipitated radiolabeled liposomes only after they had been incubated with fusogenic Sendai virions. Incubation of N-4-nitrobenzo-2-oxa-1,3-diazole-labeled fusogenic reconstituted Sendai virus particles with phosphatidylcholine/cholesterol liposomes resulted in fluorescence dequenching. Incubation of Tb3+-containing virus envelopes with phosphatidylcholine/cholesterol liposomes loaded with sodium dipicolinate resulted in the formation of the chelation complex Tb3+-dipicolinic acid, as was evident from fluorescence studies. Virus envelopes fuse efficiently also with neuraminidase/Pronase-treated erythrocyte membranes, i.e. virus receptor-depleted erythrocyte membranes, although fusion occurred only under hypotonic conditions.     

Aldehydes, hydrogen peroxide, and organic radicals as mediators of ozone toxicity

It is generally agreed that unsaturated fatty acids (UFA) are an important class of target molecule for reaction with ozone when polluted air is inhaled. Most discussions have implicated the UFA in cell membranes, but lung lining fluids also contain fatty acids that are from 20 to 40% unsaturated. Since UFA in lung lining fluids exist in a highly aquated environment, ozonation would be expected to produce aldehydes and hydrogen peroxide, rather than the Criegee ozonide. In agreement with this expectation, we find that ozonations of emulsions of fatty acids containing from one to four double bonds give one mole of H2O2 for each mole of ozone reacted. Ozonation of oleic acid emulsions and dioleoyl phosphatidyl choline gives similar results. with two moles of aldehydes and one mole of H2O2 formed per mole of ozone reacted. The net reaction that occurs when ozone reacts with pulmonary lipids is suggested to be given by equation 1. [formula: see text]. From 5 to 10% yields of Criegee ozonides also appear to be formed. In addition, a direct reaction of unknown mechanism occurs between ozone and UFA in homogeneous organic solution, in homogeneous solutions in water, in aqueous emulsions, and in lipid bilayers to give organic radicals that can be spin trapped. These radicals are suggested to be responsible for initiating lipid peroxidation of polyunsaturated fatty acids. Thus, aldehydes, hydrogen peroxide, and directly produced organic radicals are suggested to be mediators of ozone-induced pathology.     

Free radical fragmentation of cardiolipin by cytochrome c

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H₂O₂/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H₂O₂/ascorbate or with Cu²⁺/H₂O₂/ascorbate.     

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of $\text{V}$. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.