The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein

Human myelin basic protein was fractionated into its various charge isomers by CM52 cation exchange chromatography. Approximately 25-30% of the total charge applied to the column appeared in the void volume. This material termed "C-8," was further purified by reversed phase high performance liquid chromatography. Amino acid analyses of C-8 revealed low Arg (7 residue % in C-8 compared to 11-12 residue % in C-1) and increased Glx residues. The low Arg was accounted for by a corresponding amount of citrulline. Sequence analysis after chemical fragmentation (cyanogen bromide and BNPS-skatole) and enzymatic (cathepsin D and carboxypeptidase S-1) digestion localized the citrulline at residues 25, 31, 122, 130, 159, and 170 of the amino acid sequence. The effect of this loss of positive charge on the ability of the protein to aggregate lipid vesicles was demonstrated with vesicles composed of phosphatidylcholine (92.2 mol %) and phosphatidylserine (7.8 mol %). C-1 was the most effective charge isomer, and C-8 was the least effective. The ability of these charge isomers to aggregate vesicles correlated with the net positive charge on each. Vesicles composed of phosphatidylcholine alone were not aggregated by lipophilin or any of the charge isomers. However, when lipophilin was incorporated into phosphatidylcholine vesicles (50% w/w), small, optically clear suspensions of vesicles were formed. None of C-1, C-2, or C-3 aggregated these vesicles, but C-8 produced rapid vesicle aggregation. Since the substitution of citrulline for Arg would generate several relatively long apolar sequences, these would enhance the ability of C-8 to interact with the hydrophobic lipophilin molecule, promoting vesicle aggregation by hydrophobic interactions. The mechanism by which citrulline is generated in myelin is not known, although enzymatic conversion has been described in other systems. Studies are underway to elucidate the mechanism by which this post-translational modification is generated.     

Effect of bovine basic protein charge microheterogeneity on protein-induced aggregation of unilamellar vesicles containing a mixture of acidic and neutral phospholipids

Two of the charge isomers (components 1 and 2) normally found as microheteromers of myelin basic protein were isolated, and their abilities to aggregate vesicles consisting of mixed phospholipids were studied. Component 1 (the most cationic of the microheteromers) aggregated phosphatidylcholine (PC) vesicles containing 7.8 mol% phosphatidylserine (PS) more rapidly and at lower protein concentrations than component 2, which differs from component 1 by 1 net positive charge. Modification of components 1 and 2 in vitro by phosphorylation with rabbit muscle protein kinase decreased the ability of both components to aggregate vesicles. The greater the extent of phosphorylation, the less effective were the isomers at inducing aggregation. Decreasing the charge of either component 1 or component 2 by removal of the two C-terminal arginyl residues also decreased the ability of the isomers to induce aggregation. Therefore, charge microheterogeneity, whether arising in vivo or generated in vitro, markedly affected the ability of these microheteromers to aggregate PC vesicles containing 7.8 mol% PS. Because a small difference in the charge of the protein had a marked effect on vesicle aggregation, we propose that charge microheterogeneity may play an important and dynamic role in the structure and function of normal myelin.     

Secondary structure of charge isomers of myelin basic protein before and after phosphorylation

Human myelin basic protein (MBP) was fractionated into several of its charge isomers (components). Of these, the secondary structures of four isomers before and after phosphorylation have been studied by circular dichroism (CD). None of the four showed any alpha-helical structure. All of the components showed varying amounts of beta-structure, random structure, and turns. Component 1 (C-1), the most cationic of the components, showed 13%; component 2 (C-2) had 19%; C-3, 17%; and C-4, 24% of beta-structure. Each of the four components was phosphorylated with protein kinase C, from human brain. The extent of phosphorylation varied considerably from 2.8 +/- 0.6 mol of PO4/mol of protein in C-1 to 5.2 +/- 0.8 mol of PO4/mol of protein in C-4. The effect of phosphorylation on the secondary structure was to induce beta-structure in all the components. The largest change in beta-structure was in C-1 and the least in C-4. The surprising result is that although the components were phosphorylated to different extents, the amount of beta-structure in all four components increased to a final proportion of 35-40%. Treatment of phosphorylated C-1 with acid phosphatase removed 50% of the total radioactivity. Although the remainder represented approximately 1 mol of PO4/mol of protein, the proportion of beta-structure was unaltered. We concluded that a single phosphorylation site identified as residues 5-13 represented a critical size for stabilization of beta-structure of MBP in solution and that phosphorylation at the other sites had little influence on secondary structure.     

Modulation by glycosphingolipids of membrane-membrane interactions induced by myelin basic protein and melittin.

The effect of glycosphingolipids (GSLs) with oligosaccharide chains of different length and charge on membrane-membrane interactions induced by myelin basic protein (MBP) or melittin (Mel) was comparatively investigated with small unilamellar vesicles. MBP induces a fast vesicle aggregation and close membrane apposition. Merging of lipid bilayers and vesicle fusion induced by MBP are slower and less extensive processes compared to membrane apposition. The changes of membrane permeability concomitant to these phenomena are small. The Trp region of MBP remains in a rather polar environment when interacting with vesicles; its accessibility to NO3- or acrylamide quenching depends on the type of GSLs in the membrane. The Trp region of Mel is inserted more deeply into the lipid bilayer and its accessibility to the aqueous quenchers is less dependent on variations of the oligosaccharide chain of the GSLs. Mel induces a faster and more extensive membrane apposition and bilayer merging than does MBP. Extensive vesicle disruption occurs in the presence of Mel. Negatively charged GSLs facilitate membrane proximity and vesicle aggregation but an increase of the oligosaccharide chain length of either neutral or acidic GSLs decreases the interaction among vesicles that are induced by either protein. This effect is independent of the different mode of insertion of MBP and Mel into the membrane. Our results suggest that the modulation by the oligosaccharide chain on the protein-induced interactions between bilayers containing GSLs is probably exerted beyond the level of local molecular interactions between the basic proteins and the lipids.     

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.     

Barrier properties of glycophorin-phospholipid systems prepared by different methods

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K⁺:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K⁺:dextran trap = 0.67 and $\text{t}\text{1}\text{2}$ of potassium efflux at 22°C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (H_II) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.     

The role of charge microheterogeneity of human myelin basic protein in the formation of phosphatidylglycerol multilayers

Human myelin basic protein (HBP) was fractionated into its various charge isomers by chromatography on CM-52 columns at pH 10.6. Components 1,2,3 and "8" (C-1, C-2, C-3, and C-"8") were cleanly separated. Each component was combined with phosphatidylglycerol (PG) vesicles, at neutral pH at a concentration of 30% (w/w), protein/lipid. C-1, the most cationic of the components was the most effective at inducing the formation of multilayers when studied by liquid X-ray diffraction. C-3, which differs from C-1 by 2 positive charges was less effective than C-2. C-"8" was totally ineffective since the scattering pattern with this component was no different from that of the pure lipid. Thus a seemingly small change in net charge of the protein had a dramatic effect on the ability of the protein to organize the lipid into a crystalline, multilayer arrangement characteristic of compact myelin.     

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

The phosphorylation of purified phospholamban by cyclic AMP-dependent protein kinase is stimulated by phosphatidylinositol

A pure bovine phospholamban sample was phosphorylated by cyclic AMP-dependent protein kinase maximally to about 1 mol of phosphate/mol of protein (Mr 25,000), whereas phospholamban purified from bovine cardiac SR (sarcoplasmic reticulum) vesicle prephosphorylated by the protein kinase was found to contain 4.6 mol of phosphate/mol of phospholamban. The decrease in phospholamban phosphorylation occurred during the protein purification at the immunoaffinity chromatography step. The protein phosphorylation could be restored by the addition of the affinity column flow-through fraction to the phosphorylation reaction. The phosphorylation-stimulating activity of the flow-through fraction was resistant to boiling and trypsin treatment and extractable by organic solvent, suggesting that the endogenous factor(s) is lipid. Various phospholipids were found capable of stimulating the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase, but only phosphatidylinositol could stimulate the protein phosphorylation to a level achieved by the phosphorylation of SR membrane-bound phospholamban, about 5 mol of phosphate/mol. Phospholamban phosphorylated in the presence of phosphatidylinositol showed similar sites of phosphorylation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shifts as the phospholamban isolated from phosphorylated SR vesicles. Results of the present study suggest that phospholamban in SR is embedded in a phosphatidylinositol-rich microenvironment, and that this specific environment may be important for the regulation of Ca2+ pump by phospholamban.     

Substrate specificity of human plasma phospholipid transfer protein

The ability of human plasma phospholipid transfer protein to transfer L-alpha-[14C]dipalmitoylphosphatidylcholine (DPPC) from donor vesicles to acceptor high-density lipoproteins (HDL) was examined, using vesicles of different compositions and sizes, and native or chemically modified HDL. Phosphatidylcholine (PC) transfer was inhibited by both cholesterol and sphingomyelin incorporation into egg-PC vesicles. On a molar basis, cholesterol inhibited transfer about 5-fold more than sphingomyelin; however, the effects of both lipids on the fluidity of the vesicle membrane (measured by fluorescence polarization of diphenylhexatriene), were closely correlated with their effects on PC transfer activity. Increase in vesicle size, and decrease in bilayer curvature, also reduced transfer: the largest vesicles had no transfer activity at all. Addition of phosphatidic acid up to 17 mol% had no effect on PC transfer. HDL apolipoprotein lysyl residues were chemically modified by reductive methylation, citraconylation, or acetoacetylation. The effects of modification on the apolipoprotein structure and on the HDL particle were assessed by intrinsic fluorescence measurements, SDS-polyacrylamide gel electrophoresis patterns, and gel chromatography. Only acetoacetylation significantly affected any of these parameters. The ability of HDL to accept PC in the absence of phospholipid transfer protein decreased with an increase in apolipoprotein negative charge while, in the presence of phospholipid transfer protein, the acceptor ability of HDL increased up to 1.7-fold with an initial increase in negative charge and then decreased, ultimately to zero, upon extensive modification.     

Detection of surface charge-related properties in model membrane systems by aqueous two-phase partition

Unilamellar vesicles composed of phosphatidylcholine (PC) and either phosphatidic acid (PA) or phosphatidylglycerol (PG) partition to the upper poly(ethylene glycol) (PEG)-rich phase of a charge-sensitive 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 7, consistent with the vesicles bearing a net negative charge. When prepared in the presence of a pH gradient (interior acidic), PC/PA vesicles exhibit an increased partition to the top PEG-rich phase, consistent with a redistribution of the PA from the inner to the outer monolayer of the vesicle bilayer. Conversely, when prepared in the presence of a pH gradient (interior basic), PC/PG vesicles exhibit a decreased top-phase partition, consistent with a redistribution of the PG from the outer to the inner monolayer of the vesicle bilayer. Unilamellar vesicles composed of PC and stearylamine partition to the lower dextran-rich phase of a 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 8.5, consistent with the vesicles bearing a net positive charge. When prepared in the presence of a pH gradient (interior acidic), conditions under which the stearylamine is trapped on the inner monolayer of the bilayer, the vesicles now partition predominantly to the interface in a manner similar to vesicles composed of PC alone. These results demonstrate that partitioning in aqueous two-phase polymer systems is a sensitive method for monitoring the asymmetry of charged lipids in model membrane systems and also suggests that partitioning in charge-sensitive systems depends only on the physical nature of the exterior surface of the membrane.     

Interactions of oleic acid with liver fatty acid binding protein: a carbon-13 NMR study

13C NMR spectroscopy was used to probe the structural interactions between carboxyl-13C-enriched oleic acid (18:1) and rat liver fatty acid binding protein (FABP) and the partitioning of 18:1 between FABP and unilamellar phosphatidylcholine (PC) vesicles. Spectra of systems containing 2-8 mol of 18:1/mol of FABP (but no PC) exhibited one carboxyl resonance (182.2 ppm) corresponding to FABP-bound 18:1. At pH values less than 8.0, an additional carboxyl resonance, corresponding to unbound 18:1 in a lamellar phase, was observed. Both resonances exhibited ionization shifts with estimated apparent pKa values of less than 5 (bound 18:1) and greater than 7 (unbound 18:1). The intensity of the resonance corresponding to FABP-bound 18:1 increased with increasing 18:1/FABP mole ratio and at 8/1 mole ratio indicated that at least 2 and 6 mol of 18:1/mol of FABP were FABP-bound at pH 7.4 and 8.6, respectively. NMR spectra of systems containing equal concentrations (w/v) of FABP and PC and from 1 to 4 mol of total fatty acid (FA)/mol of FABP exhibited two 18:1 carboxyl resonances (182.2 and 178.5 ppm, pH 7.4). The downfield resonance corresponded to FABP-bound 18:1 and the upfield resonance to PC vesicle bound 18:1. At 1/1 mole ratio (FA/FABP), the intensities of both resonances were approximately equal, but at 4/1 mole ratio the resonance for PC vesicle bound 18:1 was 3-fold more intense than that for FABP-bound 18:1. The following conclusions are reached: (i) The carboxyl groups of 18:1 bound to liver FABP experience only one type of binding environment (the aqueous milieu adjacent to the protein surface).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of different drugs on a cytochrome c--phospholipid bilayer membrane.

Liposomes were prepared from dipalmitoyl phosphatidylcholine and dicetylphosphate and their interaction with the extrinsic membrane protein cytochrome c examined in terms of changes in 22Na permeability, electrophoretic mobility, protein binding, and motion of an incorporated spin label. The amount of cytochrome c bound displays no significant temperature dependence over the temperature range studied (from 30 to 55 degrees C) whereas cytochrome c causes an increase in 22Na efflux only above the phospholipid phase transition temperature. Interaction of the protein with the lipid vesicles causes no significant disturbance in the bilayer interior as monitored by the motion of the incorporated spin probe. The drugs 2,4-dinitrophenol and ethacrynic acid, both of which increase the magnitude of the vesicle negative charge, enhance both cytochrome c binding and its effect on 22Na permeability. In contrast, the local anesthetic dibucaine, which induces a positive surface charge on these liposomes, reduces both protein binding and the protein-induced increase in 22Na efflux. Finally, the chemicals butylated hydroxytoluene, 2-tert-butylphenol, and tert-butylbenzene, all of which cause early 'melting' of the phospholipid fatty acyl chains, block the capacity of cytochrome c to enhance 22Na permeability while having no effect on its binding to the vesicles.     

Interaction of lipoprotein lipase with phospholipid vesicles: effect on protein and lipid structure

The interaction of lipoprotein lipase (LpL) and a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), has been studied by several physical methods. Analysis of the circular dichroic spectrum of LpL gave the following fractional conformation: 35% alpha-helix, 30% beta-pleated sheet, and 45% remaining structure. No significant change in the circular dichroic spectrum of LpL was observed on addition of C14-ether-PC vesicles. The quenching of LpL fluorescence by acrylamide and iodide ion was decreased only slightly by addition of C14-ether-PC vesicles. Addition of LpL to sonicated C14-ether-PC vesicles containing entrapped carboxyfluorescein caused the release of less than 15% of the vesicle contents in 20 min, indicating that the enzyme did not disrupt the structure of the lipid. In contrast, greater than 80% of the vesicle contents were released with the addition of apolipoprotein A-I to an identical vesicle preparation. The temperature dependence of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into C14-ether-PC vesicles was not significantly altered by the addition of LpL. When LpL is added to vesicles, the bilayer structure of the vesicles is not disrupted as observed by freeze-fracture electron microscopy. However, at low ionic strength (0.1-0.25 M NaCl) significant aggregation of intact vesicles is observed by light scattering and electron microscopy. Vesicle aggregation is prevented and reversed by 1 M NaCl and by heparin. These data demonstrate that LpL binds to the surface of a lipid interface, without dramatic changes in lipid bilayer or protein structure.     

The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Phospholipid vesicle formation using nonionic detergents with low monomer solubility. Kinetic factors determine vesicle size and permeability

The method developed previously for formation of unilamellar vesicles from mixed micelles of egg lecithin and octyl glucoside [Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., & Reynolds, J. A. (1981) Biochemistry 20, 833-840] has been extended to allow for (1) use of nonionic detergents with much lower critical micelle concentrations and (2) variation in the time course of detergent removal. The results demonstrate the importance of kinetic factors, especially in the determination of vesicle size: initially formed vesicles are small, but the size increases slowly thereafter if detergent is not removed too quickly. Vesicle size remains fixed when the molar detergent/lipid ratio falls below about 1/1, and detergent removal becomes increasingly difficult thereafter, presumably because flip-flop of detergent from the inner to the outer leaflet of the bilayer membrane is very slow. Residual detergent (to about 25 mol %) has surprisingly little effect on anion permeability but increases cation permeability to the point where the normal discrimination between anions and cations (in pure lipid vesicles) is lost. Detergent added to initially detergent-free vesicles readily partitions into vesicular membranes (presumably only into the outer leaflet) and has a qualitatively similar effect on permeability. Vesicles produced by this method, regardless of residual detergent level, were found to be predominantly unilamellar: no multilamellar liposomes or other lipid aggregates could be detected within the accuracy of the methods employed.