Thermodynamic and mechanical properties of model mitochondrial membranes

Cardiolipin is a unique four-tailed, doubly negatively charged lipid found predominantly within the inner mitochondrial membrane, and is thought to be influential in determining membrane potential and permeability. To determine the role of cardiolipin in modulating the properties of membranes, this study investigates the thermodynamics of mixed cardiolipin and phosphatidylcholine monolayers and bilayers. Gibbs free energy analysis of mixed monolayers indicates that at low cardiolipin concentrations (5-10 mol%), there is a positive deviation from ideality on a pure water subphase, while at physiological salt concentrations a negative deviation from ideality is observed. The mechanical properties of bilayers containing cardiolipin were measured using micropipette aspiration. Both apparent area compressibility modulus, as well as lysis tension, decrease with increasing cardiolipin content. This destabilization indicates a decrease in the cohesive energy of the membrane. This interplay between interactions of lipids in monolayers and bilayers, suggests cardiolipin plays a dual role in modulating membrane properties. Cardiolipin enhances lateral interactions between lipids within monolayer leaflets, while simultaneously decreasing the cohesive energy of membranes at physiologically relevant concentrations. Taken together, these findings correlate with the decreased permeability and creation of folds in the inner mitochondrial membrane.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes

The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells.     

Selective binding of polymyxin B to negatively charged lipid monolayers

It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active $\text{mono}\text{-N[}{}^{14}\text{C]}\text{acetylpolymyxin B}$ derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm². Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm² of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm². These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 Ų in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10^?2 and 10^?1 M Ca²⁺ and Na⁺, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.     

Anisodamine induces the hexagonal phase in phospholipid liposomes

The effects of anisodamine on the polymorphic phase behaviour of cardiolipin and dioleoylphosphatidylcholine liposomes have been investigated by freeze-fracture electron microscopy. Anisodamine induces the formation of lipidic particles in cardiolipin liposomes at pH 7.0 and hexagonal HII tubes at pH 8.8. When the molar ratio of anisodamine and dioleoylphosphatidylcholine is 4 to 1, lipidic particles can be observed in the fracture faces.     

Membrane incorporation and induction of secondary structure of synthetic peptides corresponding to the N-terminal signal sequences of the glucitol and mannitol permeases of Escherichia coli

The 22-residue synthetic signal peptide of the glucitol permease (Enzyme IIgut of the bacterial phosphotransferase system; gut22), which in the intact protein is believed to function in envelope targeting, was found to insert into phospholipid monolayers of various phospholipid compositions up to high limiting pressures (36-41 milliNewton/m). The partition coefficient, derived from monolayer area expansion experiments, was greatest for the negatively charged gut22 when partitioning into monolayers of the zwitterionic lipid 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (about 1.1 X 10(5] as compared with that obtained with a mixture of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine and the negatively charged lipids 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol and cardiolipin. Gut22 contains a titratable histidyl residue (pKa = 6.8), and its protonation decreased the relative monolayer area increase 3-fold. Circular dichroism spectra showed that gut22 formed an amphiphilic alpha-helix when incorporated into lipid membranes (estimated percent helix = 65%). Fluorescence measurements indicated that tryptophan 11 is in a more hydrophobic environment in the presence of lipid than in its absence, with the environment being more hydrophobic at pH 5 than at pH 8. The more hydrophilic 15-residue signal peptide of the mannitol permease (mtl15) also incorporated into monolayers and detergent micelles (although to a lesser extent) with induction of secondary structure. Based on these results and a parallel with mitochondrial targeting in eucaryotes, we suggest that the induction of N-terminal amphiphilic structures and their association with a hydrophobic-hydrophilic interface are important for envelope targeting and the initiation of the membrane insertion of bacterial phosphoenol-pyruvate-dependent phosphotransferase system permeases.     

Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement.

Cardiolipin is a specific and functionally important phospholipid of mitochondria, and its biosynthesis is considered to be crucial for the assembly of this organelle. However, little information is available about the enzyme cardiolipin synthase, largely because it has not yet been isolated. We solubilized cardiolipin synthase from rat liver mitochondrial membranes with Zwittergent 3-14 and purified it by Mono Q anion exchange chromatography, Superose 12 gel filtration, and Mono P chromatofocusing. Cardiolipin synthase is one of the most acidic mitochondrial proteins (isoelectric point, pH 4-5) and appears as a 50-kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme requires CO2+ for activity, has an alkaline pH optimum (pH 8-9), and exhibits Km values of 45 and 1.6 microM for phosphatidylglycerol and CDP-diacylglycerol, respectively. Cardiolipin synthase loses activity during purification, and the activity can be partially reconstituted by the addition of phospholipids. The most effective phospholipid is phosphatidylethanolamine which reactivates in a cooperative manner. Cardiolipin reactivates hyperbolically at low concentrations but inhibits the enzyme at higher concentrations. In addition, cardiolipin shifts the sigmoidal reactivation curve of phosphatidylethanolamine toward lower concentrations. It is suggested that cardiolipin synthase requires interaction with several molecules of phosphatidylethanolamine and at least one molecule of cardiolipin for full enzymatic activity.     

Acylation of monolysocardiolipin in rat heart.

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent K(m) for oleoyl-coenzyme A and monolysocardiolipin were 12.5 microm and 138.9 microm, respectively. With linoleoyl-coenzyme A as substrate, the apparent K(m) for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 microm and 59.9 microm, respectively. Pre-incubation at 50 degrees C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [(3)H]cardiolipin was not formed from monolyso[(3)H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyltransferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A- dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.     

Asymmetric lipid bilayers Response to multivalent ions

Asymmetric lipid bilayers are formed by adjoining the hydrocarbon chains of two different lipid monolayers at the air-water interface through an aperture in a teflon partition separating two aqueous phases. It is shown that the addition of Ca²⁺ or polysine to the compartment limited by a monolayer of the neutral lipids glycerol dioleate or phosphatidylcholine results in no modification of the resistance and stability of the membrane, whereas a drastic decrease in both parameters is elicited by the presence of these ions on the opposite compartment containing a monolayer of the negatively charged cardiolipin or phosphatidylserine. The surface-charge dependence of this phenomenon indicates the persistence of the asymmetric lipid distribution in the bilayer after its formation from two different monolayers.     

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.     

心磷脂对细胞色素C氧化酶质子泵功能的影响

 用胆酸盐透析法将猪心线粒体细胞色素C氧化酶重组在含心磷脂和二肉豆寇磷脂酰胆碱的脂质体上,以还原态细胞色素C作为酶反应底物,记录脂酶体囊泡外介质液pH的变化,pH下降幅度可以反映细胞色素C氧化酶质子泵的功能。 心磷脂含量不同的细胞色素C氧化酶脂酶体质子泵功能不同。心磷脂含量在10％—40％(w/w)范围内,随心磷脂含量增高,该酶质子泵功能增强;当心磷艏含量超过50％时,该酶质子泵功能却随心磷脂含量的增加表现出下降的趋势。阿霉素可以与心磷脂紧密结合,抑制细胞色素C氧化酶的质子泵功能。然而,少量阿霉素却能增强含70％心磷脂的脂酶体的质子泵功能。     

SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.

SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.     

Identification and characterization of human cardiolipin synthase

The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.     

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.     

Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase

The selectivity of interaction of various cardiolipin analogues with beef heart cytochrome oxidase in reconstituted complexes with dimyristoylphosphatidylcholine has been studied by electron spin resonance spectroscopy, using lipids spin-labeled in the acyl chains. No difference in selectivity is observed between cardiolipin and its monolyso derivative, and similarly no selectivity is observed between phosphatidylcholine and lysophosphatidylcholine. Removal of the cardiolipin charge by methylation of the phosphate groups reduces but does not eliminate selectivity relative to phosphatidylcholine. The dependence of the lipid selectivity on head group and chain composition is in the order cardiolipin approximately equal to monolysocardiolipin greater than acylcardiolipin greater than dimethylcardiolipin greater than phosphatidylcholine approximately equal to lysophosphatidylcholine, where acylcardiolipin has the spin-label chain attached at the center -OH of the head group. The degree of association of the negatively charged cardiolipin derivatives with cytochrome oxidase decreases with increasing salt concentration, to a level comparable to that for dimethylcardiolipin. At high ionic strength there is still a marked selectivity relative to phosphatidylcholine. Li+ ions are more effective in screening the interaction than are Na+ ions, and divalent ions are more effective than monovalent ions. The selectivity for cardiolipin is only slightly reduced on titrating the protein to high pH. Alkylation of the protein with N-ethylmaleimide has little effect on the titration behavior. Covalent modification of the protein by reaction with citraconic anhydride decreases the selectivity of interaction with cardiolipin. It is concluded that cardiolipin possesses an additional specificity of interaction with cytochrome oxidase other than that of purely electrostatic origin.     

H~+诱导心磷脂的六角形Ⅱ相和H~+的跨膜转运

本文报告H~ 能诱导心磷脂由双层排列转变为六角形Ⅱ相.含心磷脂的多层脂囊泡的~(31)P中核磁共振谱显示高场峰低场肩的双层排列特点,当pH降到2时,~(31)P核磁共振谱表现为低场峰高场肩的六角形Ⅱ相特点,表明H~ 对心磷脂多形性转变的诱导作用.用oxonol-V作为探剂.H~ 可使结合在人工脂膜上的oxonl-V的吸收峰红移和光吸收增加,表明心磷脂的六角形Ⅱ相在人工脂膜上具有H~ 的载体特性,易化H~ 的跨膜转运.     

Estimation by radiation inactivation of the size of functional units governing Sendai and influenza virus fusion

The target sizes associated with fusion and hemolysis carried out by Sendai virus envelope glycoproteins were determined by radiation inactivation analysis. The target size for influenza virus mediated fusion with erythrocyte ghosts at pH 5.0 was also determined for comparison; a value of 57 +/- 15 kDa was found, indistinguishable from that reported previously for influenza-mediated fusion of cardiolipin liposomes [Gibson, S., Jung, C. Y., Takahashi, M., & Lenard, J. (1986) Biochemistry 25, 6264-6268]. Sendai-mediated fusion with erythrocyte ghosts at pH 7.0 was likewise inactivated exponentially with increasing radiation dose, yielding a target size of 60 +/- 6 kDa, a value consistent with the molecular weight of a single F-protein molecule. The inactivation curve for Sendai-mediated fusion with cardiolipin liposomes at pH 7.0, however, was more complex. Assuming a "multiple target-single hit" model, the target consisted of 2-3 units of ca. 60 kDa each. A similar target was seen if the liposomes contained 10% gangliosides or if the reaction was measured at pH 5.0, suggesting that fusion occurred by the same mechanism at high and low pH. A target size of 261 +/- 48 kDa was found for Sendai-induced hemolysis, in contrast with influenza, which had a more complex target size for this activity (Gibson et al., 1986). Sendai virus fusion thus occurs by different mechanisms depending upon the nature of the target membrane, since it is mediated by different functional units. Hemolysis is mediated by a functional unit different from that associated with erythrocyte ghost fusion or with cardiolipin liposome fusion.     

Phase diagram of mixed monolayers of stearic acid and dimyristoylphosphatidylcholine. Effect of the acid ionization

The aim of this work is to study the phase diagram of mixed monolayers composed of dimyristoylphosphatidylcholine (DMPC) and stearic acid (SA) at different ionic strength and bulk pH of the aqueous subphase. In this way, the effect of ionization of SA on the interaction and thus on phase separation with the DMPC matrix can be analyzed. To this purpose, we first determined the ionization state of pure SA monolayers as a function of the bulk subphase pH. The SA monolayers are nearly fully ionized at pH 10 and essentially neutral at pH 4 and the mixture of DMPC and SA was studied at those two pHs. We found that the DMPC-enriched phase admits more SA if the SA monolayer is in a liquid-expanded state, which is highly related to the acid ionization state, and thus to the bulk pH and ionic strength. At pH 4 the molecules hardly mix while at pH 10 the mixed monolayer with DMPC can admit between 30 and 100% of SA (depending on the lateral pressure) before phase separation is established. The addition of calcium ions to the subphase has a condensing effect on SA monolayers at all pHs and the solubility of SA in the DMPC matrix does not depend on the bulk pH in these conditions. The observed phase diagrams are independent on the manner in which the state of the mixed film is reached and may thus be considered states of apparent equilibrium.     

Drug-induced zeta potential changes in liposomes studied by laser Doppler spectroscopy

The technique of laser Doppler spectroscopy is used to measure the electrophoretic mobility of liposomes under the influence of one beta-blocking agent and three local anesthetics. All four drugs decrease the mobility (i.e., the zeta potential) of negatively charged phospholipids (soybean lipids, phosphatidylserine and cardiolipin). The mobility of electrostatically neutral pure phosphatidylcholine (zero mobility under control conditions at pH 7 and 4) is increased linearly with the logarithm of drug concentration, indicating binding and incorporation of positively charged drug molecules. The sequence of strength of activity, measured by zeta-potential changes, corresponds to that found in biological tissues: propranolol greater than tetracaine greater than lidocaine greater than procaine. For purely negatively charged lipids (phosphatidylserine, cardiolipin) the activity of the drug is higher at acidic pH, (pH 4), while for electrostatically neutral (phosphatidylcholine) or partly neutral (soybean) lipid liposomes drug activity is about the same at pH 9, 7 and 4. A Hill plot of the data reveals noncooperative drug binding. From the line width of the scattering power spectrum the mean particle radius and the average interparticle distance in the samples are determined.     

Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes.

Membrane association of cytochrome c (cyt c) was monitored by the efficiency of resonance energy transfer from a pyrene-fatty acid containing phospholipid derivative (1-palmitoyl-2[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine (PPHPC)) to the heme of cyt c. Liposomes consisted of 85 mol% egg phosphatidylcholine (egg PC), 10 mol% cardiolipin, and 5 mol% PPHPC. Cardiolipin was necessary for the membrane binding of cyt c over the pH range studied, from 4 to 7. In accordance with the electrostatic nature of the membrane association of cyt c at neutral pH both 2 mM MgCl2 and 80 mM NaCl dissociated cyt c from the vesicles completely. At neutral pH also adenine nucleotides in millimolar concentrations were able to displace cyt c from liposomes, their efficiency decreasing in the sequence ATP > ADP > AMP. In addition, both CTP and GTP were equally effective as ATP. The detachment of cyt c from liposomes by nucleotides is likely to result from a competition between cardiolipin and the nucleotides for a common binding site in cyt c. When pH was decreased to 4 there was a small yet significant increase in the apparent affinity of cyt c to cardiolipin containing liposomes. Notably, at pH 4 the above nucleotides as well as NaCl and MgCl2 were no longer able to dissociate cyt c and, on the contrary, they slightly enhanced the quenching of pyrene fluorescence by cyt c. The above results do suggest that the membrane association of cyt c at acidic pH was non-ionic and presumably due to hydrogen bonding. The pH-dependent binding of cyt c to membranes was fully reversible. Accordingly, in the presence of sufficient concentrations of either nucleotides or salts rapid detachment and membrane association of cyt c could be induced by varying pH between neutral and acidic values, respectively.