Phosphatidic acid distribution on the external surface of mixed vesicles.

A new method has been developed to detect the distribution of phosphatidic acid on the external surface of mixed phospholipid vesicles. Some positive dyes undergo large absorbance changes when the spatial separation between two or more dye molecules is smaller than a critical distance. When these dyes interact with mixed phospholipid vesicles, the absorbance changes may be utilized to calculate the amount of phosphatidic acid molecules which, on the external surface, occupy nearby positions not exceeding the critical dye distance, i.e., the amount of paired phosphatidic acid molecules. This amount was found to be higher than that calculated by statistical methods, indicating that phosphatidic acid molecules tend to be associated, in spite of the electrostatic repulsion between negative groups. The dependence of the amount of paired phosphatidic acid molecules on the pH, phosphatidylcholine:phosphatidic acid ratio, and temperature has been also analyzed.     

Interaction of complement proteins C5b-6 and C5b-7 with phospholipid vesicles: effects of phospholipid structural features

Complement components C5b-6 and C7 assemble to form C5b-7, which then interacts with membranes and commits the membrane attack complex to a target site. This protein-membrane association event was investigated to determine possible structural features that could contribute to a selective membrane attack. This system may also suggest general properties of protein-membrane insertion events. Initial binding of C5b-6 to membranes could potentially determine the site of assembly. However, binding of C5b-6 to membranes required phosphatidylglycerol or phosphatidic acid produced from egg phosphatidylcholine while binding of C5b-6 to phosphatidylcholine, phosphatidylserine, or phosphatidylinositol was undetectable. Binding to phosphatidic acid was irreversible, and the bound C5b-6 could no longer interact with C7. In contrast, C5b-7 interacted with all phospholipids tested. The rate-limiting process was the interaction of C5b-6 and C7, which displayed bimolecular properties and an activation energy of 37 kcal/mol. The C5b-7 complex showed 20-fold selectivity for small unilamellar phospholipid vesicles over large unilamellar vesicles. Vesicles carrying high negative charge densities were selected over neutral vesicles by a factor of about 5. Vesicles formed from phospholipids with short, saturated hydrocarbon side chains (dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine) were about 5-fold less effective than those formed from phospholipids with natural fatty acid distributions. The gel vs. fluid state had little influence on C5b-7 insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Phosphatidylinositol transfer protein from bovine brain: Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521–528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

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.     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.     

The flattened face of type II beta phosphatidylinositol phosphate kinase binds acidic phospholipid membranes

Type II beta phosphatidylinositol phosphate kinase is a representative phosphatidylinositol phosphate kinase that is active against membrane-bound substrates. The structure of the enzyme contains a flattened basic face that spans the crystallographic dimer interface and is adjacent to the active site. Analytical ultracentrifugation shows that phosphatidylinositol phosphate kinase is a dimer in solution. Modeling suggested that the flattened face binds to acidic phospholipids by electrostatic interactions. The enzyme binds to acidic vesicles containing phosphatidylserine, phosphatidic acid, or phosphoinositides mixed with phosphatidylcholine, but not to neutral phosphatidylcholine vesicles. Binding to acidic vesicles is abolished in the presence of 1.0 M NaCl, consistent with an essential electrostatic contribution to the free energy of binding. The +14 charge on the flattened face of the dimer was reduced to +2 in the triple mutant Lys72Glu/Lys76Glu/Lys78Glu. The mutation has no effect on dimerization, but reduces the apparent KA for 25% phosphatidylserine/75% phosphatidylcholine mixed vesicles by 16-fold. The reduction in the level of binding can be ascribed to a loss of electrostatic interactions based on the finite difference solution to the Poisson-Boltzmann equation. The mutant reduces catalytic activity toward phosphatidylinositol 5-phosphate by approximately 50-fold. The wild-type enzyme binds half-maximally to phosphatidylinositol 4,5-bisphosphate-containing vesicles at a mole fraction of 0.3% in a phosphatidylcholine background, as compared to a 22% mole fraction in phosphatidylserine. The binding to phosphatidylinositol 4,5-bisphosphate-containing membranes is less sensitive to salt and to the triple mutation than binding to phosphatidylserine-containing membranes, suggesting that at least part of phosphatidylinositol 4,5-bisphosphate's interaction with the enzyme is independent of the flattened face. It is concluded that the flattened face of type II beta phosphatidylinositol phosphate kinase binds to membranes through nonspecific interactions, and that this interaction is essential for efficient catalysis.     

Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer

Phospholipase D is used to convert egg phosphatidylcholine to phosphatidic acid in unilamellar vesicles. The transbilayer distribution of both lipids is determined by 31P NMR using paramagnetic ions. Phosphatidic acid formed in the outer monolayer is translocated to the inner monolayer with a halftime of 30-40 min or less. This is accompanied by an equally fast movement of part of the phosphatidylcholine from the inner to the outer monolayer. During these fast transbilayer movements the barrier properties of the vesicle bilayer are maintained.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Transfer properties of the bovine brain phospholipid transfer protein. Effect of charged phospholipids and of phosphatidylcholine fatty acid composition

The monolayer technique has been used to study the transfer of [¹⁴C]phosphatidylinositol from the monolayer to phosphatidylcholine vesicles. An equivalent transfer rate was found for egg phosphatidylcholine, dioleoylphosphatidylcholine, dielaidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. A reduced transfer rate was found for a shorter-chain derivative, dimyristoylphosphatidylcholine, and for species with two polyunsaturated fatty acid chains such as dilinoleoylphosphatidylcholine, diheptadecadienoylphosphatidylcholine, dilinolenoylphosphatidylcholine and diether and dialkyl derivatives. No activity was found for 1,3-dipalmitoylphosphatidylcholine. The presence of up to 5 mol% phosphatidylinositol in egg phosphatidylcholine vesicles had no effect on the transfer rate. Introduction of more than 5 mol% phosphatidylinositol or phosphatidic acid into the phosphatidylcholine vesicles gradually decreased the rate of phosphatidylinositol transfer from the monolayer. 20 mol% acidic phospholipid was nearly completely inhibitory. Transfer experiments between separate monolayers of phosphatidylcholine and phosphatidylinositol showed that the protein-bound phosphatidylcholine is readily exchanged for phosphatidylinositol, but the protein-bound phosphatidylinositol exchange for phosphatidylcholine occurs at a 20-times lower rate. The release of phosphatidylinositol is dependent on the lipid composition and the concentration of charged lipid in the acceptor membrane, but also on the ratio between donor and acceptor membranes. The main transfer protein from bovine brain which transfer phosphatidylinositol and phosphatidylcholine transfers also phosphatidylglycerol, but not phosphatidylserine or phosphatidic acid. The absence of significant changes in the surface pressure indicate that the phosphatidylinositol and phosphatidylcholine transfer is not accompanied by net mass transfer.     

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.     

Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase A1 from bovine testis

We previously purified a cytosolic phospholipase A1 that could catalyze the preferential hydrolysis of phosphatidic acid in mixed-micelle assays. Here we studied the enzyme's interactions with unilamellar lipid membranes and examined effects of the lipids on enzyme binding, stability, and catalysis. A major finding was that membrane lipids could influence the stability, activity, and specificity of the enzyme under conditions where enzyme binding to the membranes was likely to be saturated. Thus, the enzyme was unstable at 37 degrees C in the absence of membranes but bound to membranes that contained anionic phosphoglycerides and could be stabilized by these membranes in the presence of albumin. The overall activity of the bound enzyme toward membrane phosphoglycerides, assayed in the presence of albumin, increased when phosphatidylethanolamine was substituted for phosphatidylcholine. Furthermore, the enzyme's catalytic preference for phosphatidic acid increased when cholesterol and diacylglycerol were included in the membranes, sn-1-stearoyl-2-arachidonoylphosphatidylethanolamine was substituted for sn-1-palmitoyl-2-oleoylphosphatidylethanolamine, and the concentration of phosphatidic acid was increased from 0 to 10 mol % of the total membrane phosphoglycerides. Finally, changes in the relative contents of phosphatidylcholine and phosphatidylserine in the membranes influenced the enzyme's catalytic preference for different molecular species of phosphatidic acid. These results provide the first available information about the enzyme's ability to interact with membranes and identify conditions that yield high enzyme activity toward membrane-associated phosphatidic acid.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.     

Synthesis and properties of radioiodinated phospholipid analogues that spontaneously undergo vesicle-vesicle and vesicle-cell transfer

An efficient method for the synthesis and purification of a variety of iodinated phospholipid analogues is described. 1-Acyl-2-[[[3-(3-[125I]iodo-4-hydroxyphenyl)- propionyl]amino]caproyl]phosphatidylcholine (125I-PC) was prepared by alkylation of 1-acyl-2-(aminocaproyl)phosphatidylcholine with monoiodinated Bolton-Hunter reagent. 125I-Labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine were produced from 125I-PC by phospholipase D catalyzed base exchange in the presence of ethanol-amine or L-serine. All of these lipid analogues transferred readily from donor vesicles into recipient membranes. When an excess of acceptor vesicles was mixed with a population of donor vesicles containing the iodinated analogues, approximately 50% of the 125I-labeled lipids transferred to the acceptor vesicle population. In addition, under appropriate incubation conditions, these lipids were observed to transfer from vesicles to mammalian cells. Autoradiographic analysis of 125I-labeled lipids extracted from the cells after incubation with vesicles at 2 degrees C for 60 min revealed that a large proportion of the 125I-labeled phosphatidic acid was metabolized to 125I-labeled diglyceride and 125I-labeled phosphatidylcholine, whereas no metabolism of exogenously supplied 125I-labeled phosphatidylethanolamine or 125I-labeled phosphatidylcholine could be detected.     

Role of membrane lipids in the interaction of daunomycin with plasma membranes from tumor cells: implications in drug-resistance phenomena

Equilibrium binding studies on the interaction between the anthracycline daunomycin and plasma membrane fractions from daunomycin-sensitive and -resistant murine leukemia P-388 cells are presented. Drug binding constants (KS) are 15,000 and 9800 M-1 for plasma membranes from drug-sensitive and drug-resistant cells, respectively. Drug binding to the membranes is not affected by either (i) thermal denaturation of membrane proteins or (ii) proteolytic treatment with trypsin, thus suggesting that the protein components of the membranes do not have a major role in determining the observed drug binding. Also, fluorescence resonance energy transfer between tryptophan and daunomycin in the membranes indicates that interaction of protein components with the drug should not be responsible for the observed differences in drug binding exhibited by plasma membranes from drug-sensitive and -resistant cells. Plasma membranes from drug-sensitive cells contain more phosphatidylserine and slightly less cholesterol than membranes from drug-resistant cells. Differences in the content of the acidic phospholipid between the two plasma membranes seem to produce a different ionic environment at membrane surface domains, as indicated by titration of a membrane-incorporated, pH-sensitive fluorescence probe. The possible role of membrane lipids in modulating drug binding to the membranes was tested in equilibrium binding studies using model lipid vesicles made from phosphatidylcholine, phosphatidylserine, and cholesterol in different proportions. The presence of phosphatidylserine greatly increases both the affinity and the stoichiometry of daunomycin binding to model lipid vesicles. The similarity between the effects of phosphatidylserine and other negatively charged compounds such as dicetyl phosphate, cardiolipin, or phosphatidic acid suggests that electrostatic interactions are important in the observed binding of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between acetylcholine receptor function and structural properties of membranes

Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion-gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR-containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion-gating activity.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.