BILIRUBIN-PHOTOSENSITIZED LYSIS OF RESEALED ERYTHROCYTE MEMBRANES*

Abstract— The ability of bilirubin to photosensitize biomembrane damage has been tested, using resealed human erythrocyte ghosts as a model system. Continuous irradiation of bilirubin-charged ghosts with broad-band blue light resulted in membrane lysis, as evidenced by the release of trapped markers such as Na⁺ or glucose-6-phosphate. Sodium dodecyl sulfate-gel electrophoresis of membrane proteins revealed a progressive and relatively rapid loss of spectrin (bands 1 and 2) and band 4.1 during irradiation, accompanied by the appearance of cross-linked polypeptides. The antioxidant butylated hydroxytoluene protected ghosts against lysis, but not against protein cross-linking, suggesting that lipid peroxidation is the crucial membrane disrupting event.     

EFFECT OF TRANSMEMBRANE POTENTIAL ON CHARGE PHOTOSEPARATION IN LIPOSOMES

Abstract— An unexpected transmembrane potential effect on the recombination rate of the pheophytin or bacteriopheophytin anion-radicals (dissolved in membrane) and ascorbic ion-radicals (dissolved in aqueous interior) has been established in liposomes. The influence of transmembrane potential on the recombination rate of Ru³⁺ (dissolved in inner volume) and (C₁₈H₃₇)V⁺ or (C₁₄H₂₉)V⁺ (dissolved in membrane) was observed. The potential was created by a potassium concentration gradient between inner and outer volumes of liposomes in the presence of valinomycin. The effect of the potential was considered on the bases of: (1) it was determined by the diffusional drift of the hydrophobic radicals in a radial direction in the membrane, according to the direction of the electric field; (2) the electric field changed the rate constant of the electron transfer, owing to the effects on the free energy and electronic coupling. Our results show the first explanation to be preferable.     

PROOXIDANT and ANTIOXIDANT EFFECTS OF ASCORBATE ON PHOTOSENSITIZED PEROXIDATION OF LIPIDS IN ERYTHROCYTE MEMBRANES

Abstract— Continuous blue light irradiation of resealed erythrocyte ghosts at 37°C in the presence of uroporphyrin or protoporphyrin results in ¹O₂-mediated (azide inhibitable) lipid peroxidation and membrane lysis. Lipid peroxidation was assessed by thiobarbituric acid reactivity and by quantitation of total hydroperoxides, while lysis was measured in terms of trappedglucose–6-P release. Low concentrations of ascorbate, AH^- (e.g. 0.5 m M ). present at the start of irradiation, significantly enhanced the rates of lysis and peroxidation, whereas relatively high concentrations of AH^- (e.g. 15 m M ) inhibited both processes. By way of contrast. AH^- produced only a dose-dependent inhibition of the photoinactivation of lysozyme, added as an extramembranous target. No significant AH^-induced lipid peroxidation was observed in dark or light controls, plus porphyrin or minus porphyrin, respectively. Stimulation of peroxidation and lysis by low levels of AH^- was enhanced by added Fe(III), abolished by EDTA. but unaffected by catalase or superoxide dismutase. A plausible explanation for these results is as follows. At low concentrations of AH^- prooxidant activity is favored. Redox metal-mediated breakdown of photoperoxides occurs, which tends to amplify lipid peroxidation. Neither O₂^- nor H₂O₂ appears to be involved. At significantly high concentrations, AH^- acts predominantly as an antioxidant by intercepting ¹O₂ and/or sensitizer triplet, or by scavenging free radical intermediates of lipid peroxidation.     

HYPERICIN PHOTOSENSITIZATION IN AQUEOUS MODEL SYSTEMS

Abstract Photosensitization of lysozyme, liposomes, ghosts and intact red blood cells (RBC) was investigated for aqueous hypericin. The effects of azide ion, 1,4-diazabicyclo(2.2.2)octane, and superoxide dismutase on photosensitized inactivation of lysozyme in 0.5% Triton X-100 indicate that singlet oxygen is the major inactivating intermediate with a contribution from superoxide. The singlet oxygen quantum yield (Φ_Δ) scaled to methylene blue is 0.49 ± 0.06 at monochromatic wavelengths from 514 to 600 nm. Relative values of Φ_Δ based on lysozyme inactivation for different vehicles are: 0.5% Triton X-100 (1.13), human serum albumin (0.65), Cremophor-EL(0.76), Cremophor-RH40 (0.98), egg phosphatidylcholine (EPC) liposomes (0.04), hydrogenated soy phosphatidylcholine (HSPC) liposomes (<0.01). Hypericin photosensitized lipid peroxidation of EPC liposomes and RBC ghosts. Extensive cross-linking of ghost membrane proteins occurred during the initial stages of lipid peroxidation. Prompt photohemolysis was used as the assay of RBC membrane damage. The photohemolysis curves are modeled with multihit target theory based on the "hit number" (n) and the target cross section (v). The values of v and the conventional "1/t₅₀" parameter are equivalent determinants of the photohemolysis rate. The photohemolysis curves are in good agreement with n = 15 for incubation in phosphate-buffered saline at different hypericin concentrations and with additives. The measurements for other vehicles led to n = 19 for Cremophor-EL and n = 3 for EPC and HSPC liposomes, indicating that the kinetics of photohemolysis depend on the conditions of incubation.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

THE EFFECTS OF LIQUID HOLDING ON NEAR-UV(300–400 nm) IRRADIATED ESCHERICHIA COLI STRAINS DIFFERING IN NEAR AND FAR-UV RADIATION SENSITIVITY

Abstract— Stationary phase cells from four Escherichia coli strains differing in near- (nur vs. nur ₊) and far-UV (recAl vs. recA₊) radiation sensitivity were subjected to near-UV radiation (NUV) in 0.85% saline. Although the NUV-irradiated cultures yielded increased colony numbers following 24 h of liquid holding (LH), a fluctuation test for each experiment showed that the observed increases were not due to recovery but were in fact due to cell multiplication. The decline in viability observed after NUV with liquid holding using the fluctuation test was equivalent in strains RT2, 3 and 4 while the decline observed with RT1 was less marked. The discrepancy between LH involving cell densities of 10₈-10₉ and 1–4 cells/m/ can be resolved by assuming that with dense cell suspensions, NUV-induced membrane damage leads to leakage or lysis, supplying sufficient nutrients to allow growth of undamaged, surviving cells.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: STUDIES OF THE Ii700 INTERMEDIATES WITH THE PROTEIN COVALENTLY BOUND TO PREFORMED LIPOSOMES

Abstract— We have described the covalent binding of 124-kDa oat phytochrome to large unilamellar liposomes composed of either dioleoyl phosphatidylcholine or dipalmkoyl phosphatidylcholine or soybean lecithin, without affecting the photochromic properties of the protein. These phytochrome-liposome systems have now been studied by laser flash photolysis. The liposomes, independent of their membrane rigidity (liquid-crystal vs gel-like phase), do not influence the ratio and reactivity of the two primary photoproducts, Iⁱ₇₀₀- of the red absorbing form of phytochrome, P_l Thus, the lifetimes of the Iⁱ₇₀₀ intermediates and the activation parameters associated with Iⁱ₇₀₀Iⁱ_bl are the same as those measured for nonbound phytochrome in buffer solution. The temperature increase from about 273 K. to 297 K lowers the population of the shorter-lived Iⁱ₇₀₀ intermediate to the same extent both in the liposome-P_l and in nonbound P_l, whereas it does not affect the relative population of the Iⁱ₇₀₀ intermediates from non-bound P_l in the presence of 25% ethylene glycol added to the buffer solution (ionic strength 0.17).     

LIPID PEROXIDATION AND OTHER MEMBRANE DAMAGE PRODUCED IN Escherichia coli K1060 BY NEAR-UV RADIATION AND DEUTERIUM OXIDE

Abstract— With accumulating evidence that the membrane is an important site for near-UV inactivating events, we have investigated the effects of near-UV radiation on the unsaturated fatty acid auxotroph E. coli K1060 following incorporation into the membrane phospholipids of fatty acids with varying degrees of unsaturation. Sensitivity, lipid peroxidation and membrane damage, as determined by ⁸⁶Rb⁺ leakage, have been found to increase with increasing unsaturation in log-phase cells. Similar experiments carried out in D₂O also show an increase in sensitivity, lipid peroxidation and membrane damage, indicating that singlet oxygen may be responsible for such near-UV-radiation-induced damage     

Molecular interactions between phospholipids and mangostin in a lipid bilayer

Molecular interactions between phospholipids and mangostin in a lipid bilayer have been investigated in terms of the maximum additive concentration (MAC) of mangostin in liposomes, the surface potential, particle size, microscopic-viscosity and microscopic-polarity of liposomes, and the permeability of glucose. The mangostin used is a natural product extract: 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9-xanthenenone.

The MAC of mangostin was fairly dependent upon the nature of the liposomes (uncharged, negatively charged or positively charged). Solubilization of mangostin in the liposomal bilayer resulted in both an increase in the negative charge on the liposomal surface, strenghthening the state of the bilayer membrane, and a depression in the release of the glucose involved. Mangostin was found to temporarily stabilize the liposomal bilayer, although the bilayer membrane is still unstable in the long run.     

SINGLET OXYGEN GENERATION BY PORPHYRINS AND THE KINETICS OF 9,10-DIMETHYLANTHRACENE PHOTOSENSITIZATION IN LIPOSOMES

Abstract— Two new sensitizers are introduced for a potential use in photodynamic therapy: Zn²⁺- and MG²⁺-tetrabenzoporphyrin (ZnTBP and MgTBP). A comparative study of the quantum yields of singlet oxygen generation (Φ_Δ) of hematoporphyrin derivative (HpD), Photofrin II (PF-II), Zn²⁺-phthalocyanine tetrahydroxyl [ZnPC(OH)₄] and the newly introduced sensitizers ZnTBP and MgTBP in liposomes, as well as the kinetics of a photochemical reaction sensitized by them, was made by employing the fluorescent membrane probe 9,10-dimethylanthracene (DMA). We followed the photosensitization of DMA in real time by monitoring its fluorescence decrease at 457 nm and found that DMA's photosensitization is oxygen mediated. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the Φ_Δ values were evaluated. At 10 μ M sensitizer in an aqueous suspension of 2 mg/mL egg phosphatidylcholine (EPC), HpD was found to have the largest value of Φ_Δ (0.215), followed by PF-II (0.191), ZnTBP (0.023), MgTBP (0.019) and ZnPC(OH)₄ (0.005). As a test of the method, Φ_Δ for methylene blue in ethanol was measured and found to be 0.45 as compared to 0.52 reported in the literature. Due to difference in the sensitizers' absorbances at the laser's wavelength, the reaction photosensitized by ZnTBP was the fastest with a time constant of 6.7 min, followed by MgTBP (8.7), PF-II (11.9), HpD (17.1) and ZnPC(OH)₄ (31.2), all at equal sensitizers' concentrations and laser intensities. The binding constants of the sensitizers to EPC liposomes are also reported.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

THE PRODUCTION OF SINGLET MOLECULAR OXYGEN BY ZINC(II) PHTHALOCYANINE IN ETHANOL AND IN UNILAMELLAR VESICLES. CHEMICAL QUENCHING AND PHOSPHORESCENCE STUDIES

Abstract— Zn(II)phthalocyanine (ZnPc) generates O₂(¹Δ_g) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18^°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O₂(¹Δ_g) + DPBF reaction is (1.1 ±0.1) x 10⁹ M^-1 s^_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.     

A MICROSECOND KINETIC STUDY OF THE PHOTOGENERATED MEMBRANE POTENTIAL OF BACTERIORHODOPSIN WITH A FAST RESPONDING DYE

Abstract— Bacteriorhodopsin is a light activated proton pump which generates proton and electric gradients across the cytoplasmic membrane of Halobacterium halobium. In this study, a dye whose fluorescence intensity responds rapidly to membrane potential was used to follow the evolution of the potential on liposomes reconstituted with bacteriorhodopsin, in the microseconds time domain. By comparing the formation kinetics of the potential to those of the long-lived intermediate species in the bacteriorhodopsin photocycle, M₄₁₂, both in H₂O and ₂H₂O suspensions, we can draw the following conclusion: the electric potential onset time is 20 μs after initiation of the illumination. The triggering of the potential is not the formation of the M₄₁₂ intermediate, which was hitherto considered to be the first species in the bacteriorhodopsin cycle which has an unprotonated Schiff base linkage at the retinal chromophore. Rather, the potential forms at the transition of the L₅₅₀ intermediate to the species X which precedes M₄₁₂ or even at the preceding conversion of K₅₉₀ to L₅₅₀.     

Singlet-Oxygen Generation from Liposomes: A Comparison of 6β-Cholesterol Hydroperoxide Formation with Predictions from a One-Dimensional Model of Singlet-Oxygen Diffusion and Quenching

Abstract— We measured 6β-cholesterol hydroperoxide (6β-CHP), a specific singlet-oxygen (O₂(δg)) product, during irradiation of unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes containing cholesterol and zinc phthalocyanine (ZnPC). The effects of liposome size, the hydrophobic (O₂(¹δ_g)) quencher, β-carotene, and hydrophilic O₂(¹δ_g) quenchers upon the amount of 6β-CHP formed were determined and interpreted in terms of a one dimensional model of ₂(¹δ_g) quenching and diffusion. The model correctly predicted (1) that the amount of 6β-CHP was increased with increasing liposome size, (2) that P-carotene was more effective at reducing 6β-CHP formation in 400 nm diameter liposomes than 100 nm diameter liposomes and (3) that the hydrophilic quencher, water, was also more effective in large liposomes than in small liposomes.
The hydrophobic quencher, β-carotene, was more effective at reducing the formation of 6β-CHP than at reducing the 1270 nm O₂(¹δ_g) emission. This difference was found to be due to the size distribution present in the liposome preparations because the difference between the 6β-CHP data and the 1270 nm emission data was much smaller in liposome preparations with a narrow size distribution. When a significant size distribution was present, the 6β-CHP data were weighted more heavily with large-diameter liposomes, while the 1270 nm emission data were weighted more heavily with small-diameter liposomes.     

THE EFFECT OF PROTOPORPHYRIN ON HISTAMINE SECRETION BY RAT PERITONEAL MAST CELLS: A DUAL PHOTOTOXIC REACTION

Abstract— Protoporphyrin-induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80-stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100ng/m/ (0.17 μM), histamine secretion was completely inhibited after 30min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible, however, it did not develop into cell lysis. More severe phototoxic reactions in mast cells could not be produced by increasing the PP concentration or the incubation time; however, cell lysis was evoked by increasing the light intensity between 180–950W/m², using a light source with emission maxima in the 350–470nm region. Dual phototoxic effects could also be demonstrated in erythrocytes by manipulating the illumination conditions. Increased resistance to osmotic lysis was seen under moderate conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin-phototoxicity, although the light intensities required were higher both for inhibition (60–130W/m²) and lysis (280–950W/m²). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350–470nm region.     

Silicone-stabilized liposomes

The present work is focused on stabilization of liposomes by covering their surface with a thin silicone layer. The appropriate silicone monomer was obtained by the hydrosilylation of vinylmethyldimethoxysilane with 1,3,5,7-tetramethylcyclotetrasiloxane. The surface potential of egg yolk phosphatidylcholine vesicles was modified by incorporation of a cationic double-tailed surfactant, dimethyldioctadecylammonium bromide (DODAB), yielding cationic liposomes. The silicone material was deposited on the cationic liposomes in base-catalyzed polycondensation/polymerization processes of the monomer at the liposomal surface. In order to initialize the processes pH of the liposomal dispersion was adjusted to the values of 8.5 or 10.2. The formed structures were characterized using dynamic light scattering (DLS) and zeta potential measurements. The DLS measurements show that the size of covered liposomes decrease during the reaction and the zeta potential turned to negative value, as can be expected. The morphology of the structures was evaluated using transmission cryo-electron microscopy (cryo-TEM). The cryo-TEM micrographs revealed the presence of the covered liposomes of sizes lower than the initial liposomes, which is in line with DLS measurements. However, some disintegration of the liposomes occurred during the covering procedure, especially at high pH value. Using the surfactant lysis and calcein-release study it was shown that silicone-covered liposomes are stable.     

Long-circulating gadolinium-loaded liposomes: potential use for magnetic resonance imaging of the blood pool

In our previous paper, we reported a method of liposome loading with Gadolinium (Gd) via so called polychelating amphiphilic polymer (PAP). A novel Gd-containing polymeric probe, suitable for the incorporation into the liposomal membrane, was prepared from a low-molecular-weight DTPA-polylysine by linking its N-terminus to a lipid anchor, NGPE-PE. When compared with known membranotropic MR probes, such as Gd-DTPA-SA and Gd-DTPA-PE, liposomes containing new membrane-bound polychelator possess enhanced relaxivity for water protons resulting in an increase of tissue signal intensity on MR images. In this study, we developed the optimized protocol to prepare a liposomal MR contrast agent with high relaxivity and narrow size distribution. Gd-containing liposomes were additionally modified with PEG to provide longevity in vivo. We also demonstrated that upon intravenous administration in rabbit and dog, the new preparation causes a prolonged decrease in the blood T₁ value (reflecting the proton relaxation rate in the blood) and may be considered as a potential contrast agent for MRI of the blood pool.     

Effect of variation in the chain length and number in modulating the interaction of an immunogenic lipopeptide with biomembrane models

A differential scanning calorimetry study was carried out to investigate the effect exerted by immunogenic synthetic lipopeptides obtained by the conjugation of LCMV_33–41 peptide with lipoamino acids (Laas) bearing different alkyl chain lengths (C₁₂ and C₁₆) and number of chains (2 × C₁₂) on the thermotropic behaviour of dimyristoylphosphatidylcholine (DMPC) liposomes. The aim of this work was to study the ability of these compounds to be carried by a liposomal system and released to a biomembrane model.

The examined compounds caused variations of the thermotropic parameters that characterise the liposomal system (transition temperature, T_m and enthalpy variation, ΔH), and interacted with the biomembrane models in different way. The interaction was found to be modulated by the length and number of chains present in the examined compounds. In fact, the compounds with higher number of lipid chain showed a stronger interaction with the biomembrane models with respect to the pure peptide and the compounds with a single lipid chain. These results suggest that the lipoamino acid moiety could favour the peptide to be carried by the liposomal system and released to biomembrane.     

Effect of host lattice on antigenicity of glycophorin in membranes

The influence of the host lattice on the antigenicity of glycophorin in membranes was confirmed by complement-dependent immune lysis of liposomes with two rabbit antisera, which were prepared by immunization with either human red blood cells or isolated glycophorin A. The immune lysis by either antiserum depended on the kind of phospholipid in the liposomes. Anti-glycophorin antiserum more strongly recognized glycophorin in egg-lecithin membranes than in dipalmitoyl-lecithin membranes, as did anti-red blood cell antiserum. Cholesterol in the liposomal membranes influenced the antigenicity of glycophorin. The relationship between the state of glycophorin in membranes and recognition by antibody is discussed.