The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.     

The hydrolysis of monolayers of phosphatidyl-[Me-14C]choline by phospholipase D

1. The hydrolysis of monolayers of phosphatidyl[Me-(14)C]choline at the air/water interface by phospholipase D (phosphatidylcholine phosphatidohydrolase) was investigated by a surface-radioactivity technique by using a flow counter. 2. Phosphatidylcholine of high specific radioactivity was prepared biosynthetically in good yield from [Me-(14)C]choline by using Saccharomyces cerevisiae. 3. At initial monolayer pressures between 12 and 25 dynes/cm. the hydrolysis occurred in two stages, an initial slow hydrolysis followed by a rapid hydrolysis. Below 3dynes/cm. and above 28dynes/cm. no enzymic hydrolysis of pure phosphatidylcholine monolayers could be detected. 4. The rapid hydrolysis was proportional to the enzyme concentration in the subphase, its pH optimum was 6.6, and 0.2mm-Ca(2+) was required for maximal activity. 5. Hydrolysis of the film was accompanied by a pronounced fall in the surface pressure even though the phosphatidic acid formed did not leave the film. When the pressure fell to low values the hydrolysis ceased even if the film was only partially hydrolysed. 6. Above monolayer pressures of 28dynes/cm. enzymic hydrolysis could be initiated by inclusion of phosphatidic acid (and less effectively stearyl hydrogen sulphate) in the film, although the rates were not appreciably higher than those observed at 25dynes/cm. with a pure phosphatidylcholine film. 7. The initiation of the hydrolysis by phosphatidic acid was facilitated by the inclusion of high Ca(2+) concentrations and certain carboxylic acid buffer anions in the subphase, although these did not activate by themselves. 8. The initiation of the hydrolysis at high pressures could not be related to any change in the surface potential brought about by the addition of the long-chain anions to the film, nor could it be ascribed to a surface dilution effect. 9. The results are discussed in relation to previous studies on the hydrolysis of phosphatidylcholine particles by the enzyme and also similar investigations on phosphatidylcholine monolayers with other phospholipases.     

Specific interaction of concanavalin a with glycolipid monolayers

The effect of ¹³¹I-labelled concanavalin A on the surface pressure and surface radioactivity of monolayers formed from phospholipids and from natural and synthetic glycolipids has been studied. The lectin binds to and penetrates dipalmitoyl phosphatidylcholine monolayers at a surface pressure of 15 dynes/cm and this interaction is inhibited by the presence of α-methyl mannose int he subphase. At surface pressures of 25 dynes/cm or higher, concanavalin A will interact with monoglucosyl diglyceride or diglucosyl diglyceride from Acholeplasma laidlawii and with synthetic glycolipids containing $\text{2}\text{or}\text{3 α1 → 4-}\text{linked}$ D-glucose residues in the headgroup, but not with phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or with the ganglioside II³NeuAc-GgOse₄-Cer. The binding to the glycolipid sugar group and penetration of the hydrocarbon region seem to occur simultaneously, as the time courses for the development of surface pressure and surface radioactivity coincide.     

The significance of cytochrome c redistribution during the subcellular fractionation of rat liver

1. The redistribution of mitochondrial cytochrome c during homogenization and subcellular fractionation of the liver was studied. Chromatographically homogeneous (14)C-labelled cytochrome c was added in different amounts to liver suspensions immediately before homogenization and the adsorption of radioactivity was determined in cytochrome c fractions extracted at pH4.0, first with water and then with 0.15m-sodium chloride. 2. The soluble cytochrome c remaining in the cell sap after subcellular fractionation was 7% of the calculated amount of cytochrome c passing through a soluble form during the whole process. The total amount of cytochrome c released in a soluble form and subsequently redistributed was 25-30% of the total liver cytochrome c. 3. In the standard microsomal fraction the cytochrome c extracted with water originated entirely from redistribution whereas that extracted with 0.15m-sodium chloride was 80% endogenous. In the mitochondrial fraction both cytochrome c pools were truly endogenous, so that practically none of the mitochondrial cytochrome c released to the soluble cell sap was readsorbed by the mitochondria. 4. These results support our former hypothesis that the cytochrome c extracted with 0.15m-sodium chloride at pH4.0 from the standard microsomes represents the cytochrome c newly synthesized in situ, since it does not originate from redistribution. However, the microsomal pool extracted with water cannot be an intermediate in the postulated transfer of cytochrome c from the microsomal particles to the mitochondria, since this pool arises from redistribution of mitochondrial cytochrome c.     

The biosynthesis of cytochrome c. Sequence of incorporation in vivo of [14C]lysine into cytochrome c and total proteins of rat-liver subcellular fractions

1. In order to determine the initial intracellular site of synthesis of cytochrome c in the liver cell, groups of rats were injected with [(14)C]lysine and killed 7.5, 15, 30 and 60min. later. The livers were homogenized in 0.3m-sucrose and subcellular fractions obtained. The mitochondrial fraction was further subfractionated. Pure cytochrome c was isolated from extracts of each fraction, obtained first with water at pH4.0 and then with 0.15m-sodium chloride. 2. A comparison of the kinetics of incorporation of [(14)C]lysine into total protein for each particulate fraction showed the usual two different kinds of kinetics. Incorporation into all the mitochondrial subfractions and the nuclear fraction rose gradually to a plateau value at about 20min., in contrast with that into the two microsomal fractions which rose rapidly to a peak value about seven times that for the mitochondrial fractions. The kinetics for the incorporation into mitochondrial cytochrome c showed a plateau value at 30min. about three times that for the total mitochondrial protein. There was no difference in the specific radioactivity of the mitochondrial cytochrome c extracted with water or 0.15m-sodium chloride or between the different mitochondrial subfractions. In contrast, the cytochrome c isolated from water extracts of the microsomal fractions had a lower specific radioactivity than that obtained from the 0.15m-sodium chloride extract. The specific radioactivity of the latter showed a rapid rise to a peak value about four times that for the mitochondrial cytochrome c, and the shape of the curve was similar to that for the total protein of the microsomal fraction. The results suggest that cytochrome c is synthesized in toto by the morphological components of the microsomal fraction. It seems first to be bound tightly to a microsomal particle, passing then to a looser microsomal binding and being finally transferred to the mitochondria. The newly synthesized cytochrome c in the mitochondrion could not be differentiated from the old by its degree of extractability at pH 4.0.     

Stability of unimolecular films of 32P-labelled lecithin

1. The stability of monolayers of a highly unsaturated yeast lecithin labelled with (32)P has been investigated by a surface radioactivity technique. 2. Lecithin films on distilled water at all surface pressures between 6 and 48dynes/cm. were completely stable on rapid perfusion of the subphase and on addition of ionic amphipathic substances to the film. 3. Ultrasonically treated lecithin added to the subphase caused a slow loss of surface radioactivity but little pressure change. 4. The addition of proteins to the subphase caused negligible changes in the film even when conditions were favourable for electrostatic heterocoagulation and penetration. 5. Lecithin films were not hydrolysed by a strongly acid subphase at room temperature. The very low rate of hydrolysis produced by alkali was proportional to the subphase OH(-)ion concentration: the apparent activation energy and temperature coefficient (Q(10)) of the reaction were 14250 cal. and 2.37 respectively. 6. Alkaline hydrolysis of lecithin monolayers was markedly stimulated by adding methanol (10-20%, v/v) to the subphase. The addition of ionic amphipaths to the monolayer had the expected type of effect on the hydrolysis rate, but its magnitude was far less than that suggested by an application of the Poisson-Boltzmann equation for ion distribution at a charged interface (Davies & Rideal, 1963).     

Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I' band of the protein and the CH(2) and CD(2) stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.     

Changes of the phospholipid bilayer structure under the adsorption of ferricytochrome c on its surface]

Changes in the mobility of phospholipid molecules in liposomes membranes under adsorption ferricytochrome c on its surface were studied by means of NMR and EPR spectroscopy. It is found that the interaction of cytochrome molecules with vesicles causes the broadening of 1H-NMR signals of hydrophobic as well as polar groups in cardiolipin and phosphatidylcholine in the presence of lauric or phosphatidic acid. This broadening of 1H-NMR signals in hydrophobic groups may be caused by decrease in the rate of lateral diffusion of phospholipid molecules. The changes in the correlation time of hydrophobic spin-proub in liposomes containing phosphatydiloholine and cardiolipin with the increase of ferricytochrome c concentration were also observed. These changes suggest that the formation of protein-phospholipid clusters results in the impair of the regular structure of phospholipid bilayer.     

The penetration of serum albumin into phospholipid monolayers of different fatty acid chain length and interfacial charge

1. The highest surface pressure of phosphatidylcholine monolayers allowing penetration of delipidated serum albumin decreased in the order dibehenoyl>distearoyl>dipalmitoyl=dimyristoyl. This pressure was not related to the area occupied or to the space available between the phospholipid molecules at the interface. 2. Penetration of albumin into yeast phosphatidylcholine monolayers was increased by adding a small percentage of long-chain anions (phosphatidic acid, dicetylphosphoric acid) to the film but only when the protein was below its isoelectric point (i.e. positively charged). 3. Stearylamine added to phosphatidylcholine monolayers had no effect on albumin penetration even when the protein was oppositely charged to that of the phospholipid/water interface. 4. The results are discussed in relation to the activation of certain phospholipases by anionic amphipathic substances.     

An analysis of the interaction of protein with lipid monolayers at the air/water interface

1. Measurements have been made of the interaction of cytochrome c, bovine serum albumin and synthetic oxytocin with low-pressure (2dyn/cm) monolayers of stearic acid, phosphatidylcholine and phosphatidylethanolamine. 2. [(14)C]Carboxymethylation of the cytochrome c and albumin followed by surface-radioactivity determinations have shown that only a proportion of the protein added to the subphase is bound to the monolayers and that initially the degree of binding is dependent on the protein concentration. The binding is irreversible in the sense that the adsorbed protein cannot be removed by transferring the film containing the interacted protein to a fresh subphase containing no protein. 3. Three successive types of interaction can usually be recognized. (a) Initially, whole molecules of protein penetrate the lipid film and occupy the same area as those of the protein spread at the air/water interface. (b) Above certain film pressures a part of each protein molecule, probably hydrophobic side chains, penetrates the film. The change in surface pressure per unit of bound protein is much smaller than in (a). (c) At higher film pressures, adsorption without penetration occurs. With cytochrome c this is initially dependent on a favourable electrostatic interaction.     

Shielding of phospholipid monolayers from phospholipase C hydrolysis by alpha-lactalbumin adsorption

α-Lactalbumin interacts more strongly with lecithin and cardiolipin monolayers at pH 3～4 than at pH 7 to 10. At physiological pH this protein does not penetrate monolayers of DPPC and cardiolipin above pressures of 30 dynes/cm. Enzymatic hydrolysis of these monolayers by phospholipase C (Clostridium Welchii) is inhibited partially or totally when α-lactalbumin is injected in the subphase prior to the enzyme injection.     

Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.     

Incorporation of a synthetic mitochondrial signal peptide into charged and uncharged phospholipid monolayers

The interaction of the chemically synthesized 25-residue signal peptide of subunit IV of yeast cytochrome c oxidase with synthetic and natural phospholipids was studied by using a monolayer technique. Incorporation of the peptide into phospholipid monolayers was measured as surface area increase at constant surface pressure. The peptide was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into phospholipid monolayers up to limiting pressures of 30-40 mN/m. The incorporation of the positively charged peptide was strongly enhanced by the presence of negatively charged phospholipids. The molecular area of the signal peptide in monolayers was determined with a 14C-labeled signal peptide and was 560 +/- 170 A2. This is consistent with a 25-residue alpha-helical peptide incorporating with its long axis parallel to the plane of the monolayer. Incorporation isotherms into synthetic phosphatidylcholine and phosphatidylglycerol monolayers at different charge densities were analyzed in terms of a simple incorporation/binding model, involving partitioning of the peptide into the monolayer and an in-plane binding reaction of the negatively charged phospholipids to the partitioned peptide.     

Effects of lipid composition and packing on the adsorption of apolipoprotein A-I to lipid monolayers

To better understand the factors controlling the binding of apolipoprotein molecules at the surfaces of serum lipoprotein particles, the adsorption of human apolipoprotein A-I to phospholipid monolayers has been studied. The influence of lipid packing was investigated by spreading the monolayers at various initial surface pressures (pi i) and by using various types of lipid. The adsorption of 14C-methylated apolipoprotein A-I was monitored by simultaneously following the surface radioactivity (which could be converted to the surface concentration of protein, gamma) and the change in surface pressure (delta pi). In general, increasing the pi i of lipid monolayers reduces the adsorption of apolipoprotein A-I; for expanded egg phosphatidylcholine (PC) monolayers at pi i greater than or equal to 32 dyn/cm, gamma and delta pi are zero. The degree of adsorption of the apolipoprotein is also influenced by the physical state of the lipid monolayers. Thus, at a given pi i, apolipoprotein A-I adsorbs more to expanded monolayers than to condensed monolayers so that, at a given subphase concentration of protein, gamma of apolipoprotein A-I with various phospholipid monolayers decreases in the order egg PC greater than egg sphingomyelin greater than distearoyl-PC. The plot of gamma against pi i for adsorption of apolipoprotein A-I to dipalmitoylphosphatidylcholine (DPPC) monolayers shows an inflection at pi i = 8 dyn/cm; at this pi, the DPPC monolayer undergoes a phase transition from liquid (expanded) to solid (condensed) state. Addition of cholesterol generally decreases the adsorption of apolipoprotein A-I to egg PC monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amphotericin B-sterol complex formation and competition with egg phosphatidylcholine: a monolayer study

The conditions of formation of amphotericin B-cholesterol or -ergosterol complexes in monolayers are investigated by the penetration into a monolayer of egg phosphatidylcholine/sterol of 14C-labelled N-fructosyl-amphotericin B dissolved in the aqueous subphase. An increase of both surface pressure and radioactivity as a function of concentration are observed simultaneously while a 'saturation' effect occurs only for the surface pressure. The experiments are not accurate enough to make conclusions about the number of actually penetrated amphotericin B molecules. Therefore, the existence of an amphotericin B-sterol complex was evidenced from a study of surface pressure area per molecule isotherm. The results indicate that a complex with a 2:1 stoichiometry is formed and that the amphotericin B-ergosterol interaction is larger than the amphotericin B-cholesterol interaction. The complex is dissociated by addition of egg phosphatidylcholine due to a competition between egg phosphatidylcholine and amphotericin B for sterol.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.

The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.     

Activation of protein kinase C in lipid monolayers

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.     

Mechanism of activation of cytochrome c peroxidase activity by cardiolipin

In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe...S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695-700 nm (A(695)) were compared. A strict concurrency between Fe...S(Met80) breaking (A(695)) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0 : 1 to 50 : 1 (by chemiluminescence). Nevertheless, when A(695) completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (=4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16-20-fold. A(695) did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1 : 10 : 10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe...S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe...S(Met80) breaking as the result of conformational changes in the protein globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H(2)O(2) as a result of the hydrophobic interaction between cardiolipin and cytochrome.     

Interaction of plasma apolipoproteins with lipid monolayers.

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...     

Fusion of phospholipid vesicles mediated by cytochrome c

Fusion of phosphatidylserine/phosphatidylethanolamine (1/1) vesicles induced by cytochrome c is studied at a wide range of pH values. A pH profile for the fusion with maximum values at pH 5 and pH 8 is obtained and this is found to be similar to the profile for cytochrome c binding to the vesicles. The binding property of apocytochrome c to the same phospholipid vesicles is found to be about the same as that of the cytochrome c at low ionic strength, but very different at high salt concentrations. No appreciable fusion of vesicles by apocytochrome c is observed. Proteolytic treatment and dansyl chloride labeling of cytochrome c- and apocytochrome c-vesicle complexes show that the C-terminal segments of these proteins with molecular weights of about 3000 and 5000, respectively, penetrate the bilayer. The hydrophobic labeling studies with photoreactive phosphatidylcholine in the bilayer show that segments of both cytochrome c and apocytochrome c go deep into the bilayer.