Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.     

Effect of Lipid Structure on the Capacity of Myelin Basic Protein to Alter Vesicle Properties: Potent Effects of Aliphatic Aldehydes in Promoting Basic Protein-Induced Vesicle Aggregation

The capacity of myelin basic protein or of poly-L-lysine to promote leakage of carboxyfluorescein from vesicles or the aggregation of vesicles was studied. The vesicles were composed of phosphatidylcholine as the sole or major lipid component. Addition of 10% sphingomyelin, 10% phosphatidylglycerol, 10% egg or bovine brain phosphatidylethanolamine, or 30% dodecanal had relatively little effect on the extent of carboxyfluorescein release in the presence of either myelin basic protein or poly-L-lysine. In contrast with these results, the extent of vesicle aggregation was very sensitive to lipid composition. Addition of 10% phosphatidylglycerol induced more aggregation than the other phospholipids tested. Admixing 10% of a partially degraded sample of bovine brain phosphatidylethanolamine also led to a large amount of aggregation induced by the myelin basic protein. This latter aggregation appeared more specific for the basic protein, as it occurred to a much smaller extent with poly-L-lysine. In general, the effects of the myelin basic protein on either carboxyfluorescein release or vesicle aggregation were similar to, although somewhat greater than, that of poly-L-lysine. The aggregation of vesicles containing degradation products of phosphatidylethanolamine can be ascribed largely to the presence of aliphatic aldehydes. The effect of aliphatic aldehydes was specific in that the aliphatic alcohol, hexadecanol, or the short-chain aldehydes, acetaldehyde or butyraldehyde, did not promote myelin basic protein-induced vesicle aggregation. In addition, poly-L-lysine was less effective than the basic protein in aggregating vesicles containing aliphatic aldehydes. (ABSTRACT TRUNCATED AT 250 WORDS)     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic proteins

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca2+ in the micromolar range. Optimal activity is reached at a free Ca2+ concentration of about 2 microM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with 45Ca2+, 86Rb+, or 22Na+, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80--90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb+ and Na+ was measured, when the vesicles were pretreated with ATP and Mg2+.     

Preparation and characterization of unilamellar myelin vesicles

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.     

Aliphatic aldehydes promote myelin basic protein-induced fusion of phospholipid vesicles

Myelin basic protein induces slow and limited fusion of phospholipid vesicles composed of a mixture of phosphatidylcholine and phosphatidylethanolamine. Addition of palmitoyl aldehyde to these vesicles dramatically increases their ability to fuse in the presence of myelin basic protein. Compared to aliphatic aldehydes, fatty acids are much less potent promoters of myelin basic protein-induced membrane fusion. The ability of aliphatic aldehydes to promote myelin basic protein-induced membrane fusion may be of relevance to myelin structure and function and, particularly, to the pathology of demyelinating diseases such as multiple sclerosis.     

Aliphatic aldehydes promote myelin basic protein-induced fusion of phospholipid vesicles

Myelin basic protein induces slow and limited fusion of phospholipid vesicles composed of a mixture of phosphatidylcholine and phosphatidylethanolamine. Addition of palmitoyl aldehyde to these vesicles dramatically increases their ability to fuse in the presence of myelin basic protein. Compared to aliphatic aldehydes, fatty acids are much less potent promoters of myelin basic protein-induced membrane fusion. The ability of aliphatic aldehydes to promote myelin basic protein-induced membrane fusion may be of relevance to myelin structure and function and, particularly, to the pathology of demyelinating diseases such as multiple sclerosis.     

Stopped-flow studies of myelin basic protein association with phospholipid vesicles and subsequent vesicle aggregation

When mixed with vesicles containing acidic phospholipids, myelin basic protein causes vesicle aggregation. The kinetics of this vesicle cross-linking by myelin basic protein was investigated by using stopped-flow light scattering. The process was highly cooperative, requiring about 20 protein molecules per vesicle to produce a measurable aggregation rate and about 35 protein molecules per vesicle to produce the maximum rate. The maximum aggregation rate constant approached the theoretical vesicle-vesicle collisional rate constant. Vesicle aggregation was second order in vesicle concentration and was much slower than protein-vesicle interaction. The highest myelin basic protein concentration used here did not inhibit vesicle aggregation, indicating that vesicle cross-linking occurred through protein-protein interactions. In contrast, poly(L-lysine)-induced vesicle aggregation was easily inhibited by increasing peptide concentrations, indicating that it did cross-link vesicles as a peptide monomer. The myelin basic protein:vesicle stoichiometry required for aggregation and the low affinity for protein dimerization suggested that multiple protein cross-links were needed to form a stable aggregate. Stopped-flow fluorescence was used to estimate the kinetics of myelin basic protein-vesicle binding. The half-times obtained suggested a rate constant that approached the theoretical protein-vesicle collisional rate constant.     

Modulation of myelin basic protein-induced aggregation and fusion of liposomes by cholesterol, aliphatic aldehydes and alkanes

The effect of cholesterol on myelin basic protein-induced aggregation of zwitterionic phospholipid vesicles was studied by turbidimetry, quasi-elastic light scattering and centrifugation techniques. Without cholesterol, the degree of vesicle aggregation caused by myelin basic protein is relatively low and is only slightly increased using cholesterol concentrations up to approx. 25-30 mol%. When the cholesterol content in the bilayer exceeds approx. 30 mol%, there is a dramatic increase in the susceptibility of the vesicles to aggregation in the presence of myelin basic protein. Palmitoyl aldehyde and eicosane, substances resembling products of lipid degradation, increase myelin basic protein promoted fusion of vesicles. The fusion is accompanied by increased leakage of entrapped carboxyfluorescein. In the presence of cholesterol, myelin basic protein-induced fusion of the liposomes becomes much more sensitive to the presence of aliphatic aldehydes or alkanes. The results suggest that cholesterol has an important role in promoting membrane adhesion in biological systems but these structures become unstable in the presence of small amounts of products of lipid degradation. The findings have important implications to the understanding of the stability of the myelin membrane.     

Interaction and fusion of unilamellar vesicles containing cerebrosides and sulfatides induced by myelin basic protein

The effects of myelin basic protein on the aggregation, lipid bilayer merging, intercommunication of aqueous compartments and leakage of small unilamellar vesicles of egg phosphatidylcholine containing different proportions of galactocerebroside and sulfatide were investigated. This was performed employing light scattering, absorbance changes and fluorescence assays (resonance energy transfer, Terbium/dipicolinic acid assay and carboxyfluorescein release). The apposition of membranes rapidly induced by myelin basic protein is enhanced by sulfatide but reduced by galactocerebroside compared to vesicles of egg phosphatidylcholine alone. On the other hand, the presence of either glycosphingolipid in the membrane interferes with the induction by myelin basic protein of lipid bilayer merging, subsequent fusion and changes of the membrane permeability. Our results support an important modulation by sulfatide and galactocerebroside on the interactions among membranes induced by myelin basic protein, depending on the relative proportions of the glycosphingolipids and phosphatidylcholine.     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic protein

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca²⁺ in the micromolar range. Optimal activity is reached at a free Ca²⁺ concentration of about 2 μM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with ⁴⁵Ca²⁺, ⁸⁶Rb⁺, or ²²Na⁺, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80–90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb⁺ and Na⁺ was measured, when the vesicles were pretreated with ATP and Mg²⁺.     

Mechanism of the interaction between myelin basic protein and the myelin membrane; the role of arginine methylation

The addition of solutions of bovine myelin basic protein to suspensions of unilamellar vesicles prepared from whole myelin suspensions results in the rapid equilibrium association of the vesicles into dimers, followed by time-dependent aggregation reactions. Other cationic proteins also induce the dimerization of the vesicles and equilibrium constants for dimer formation are obtained for bovine myelin basic protein, lysozyme, polyhistidine and myelin basic protein from carp, which differs from the bovine protein in that it contains no methylarginine residues. The bovine protein is more efficient at inducing dimer formation than the carp protein by approximately 0.93 kcal/mole; the carp protein is approximately as effective as the other cationic proteins examined. Complete methylation of the bovine MBP by AdoMet:MBP methyltransferase increases the interaction between MBP and the membrane by approximately 0.13 kcal/mole, consistent with the suggestion that a large portion of the free energy difference between the carp and bovine proteins arises from favorable interactions involving the methylarginine residues.     

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

Biosynthesis of the Myelin 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterases

We have investigated the site of synthesis of the 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin-associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane-bound receptor.     

Immune lysis of reconstituted myelin basic protein--lipid vesicles and myelin vesicles

Complement-mediated lysis of reconstituted lipid-myelin basic protein (BP) vesicles and myelin vesicles due to antibody raised against BP and isolated myelin is measured by determination of the amount of a water-soluble spin label, tempocholine chloride, released from the vesicles. The response is shown to be antigen-specific, antibody-dependent, and complement mediated. The relative response to different anti-BP antibody samples is similar to that determined by radioimmunoassay procedures. In contrast to immunoassays with BP in aqueous solution, this method measures immune recognition of the protein in either a synthetic or a natural membranous environment. This is important because this protein has been shown to have a different conformation when bound to lipid bilayers than in aqueous solution and its conformation depends on lipid composition. It is also a more rapid method because no separation of spin label still trapped in the vesicles and that released due to immune lysis is required. In synthetic membranes consisting of sphingomyelin, cholesterol, and an acidic lipid, either phosphatidylglycerol, phosphatidic acid, or phosphatidylserine, the response was greatest when the acidic lipid was phosphatidic acid. The response did not depend significantly on the antigen concentration expressed as molar ratio of BP to sphingomyelin, over the range 0.15:600 to 2:600, although it decreased at molar ratios less than 0.15:600. The antigen density required for immune lysis of vesicles containing this protein antigen is similar to that reported elsewhere for lipid antigens, although the time required for maximal lysis was greater. Both anti-BP and anti-myelin antibodies caused a greater specific complement-mediated response with synthetic vesicles than with myelin vesicles, which may be due to the different lipid and/or protein composition of myelin. Response was also obtained with the myelin vesicles, however, indicating that some determinants of BP can be recognized on the surface of the bilayer in isolated myelin by anti-BP.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein: a possible role in myelin formation.

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, L-alpha-dimyristoyl phosphatidylcholine and DL-alpha-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15-18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associated species. A similar interaction was observed with diacyl phosphatidylserine vesicles. These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myelin, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Spontaneous vesicularization of myelin lipids is counteracted by myelin basic protein

Hand-vortexed dispersions of several lipids (cerebrosides, sulfatides, PC, PE, PS and sphingomyelin), mixed in the ratios found for these categories of lipids in myelin, exhibit 31P-NMR spectra which have contributions from both isotropic and lamellar resonances. Investigation of this system by freeze-fracture electron microscopy and X-ray diffraction revealed that this lipid mixture has spontaneously formed small unilamellar vesicles (SUVs) (diam. approximately 400 A) and large highly convoluted unilamellar vesicles (LUVs) (diam. approximately 1000 A), the latter possibly resulting from aggregation and fusion of the SUV structures. This vesicularization of the myelin lipids was reversed by the addition of myelin basic protein: only large multilamellar aggregates were formed in the presence of protein, as shown by all three experimental methods. Although no rigorous physical-chemical explanation for these phenomena is yet available, the possibility is suggested that the high concentration of cerebrosides and/or phosphatidylethanolamine in this particular mixture of myelin lipids play pivotal roles in the formation of these unusual vesicles. Spontaneous vesicularization of myelin lipids is discussed as a potential pathway toward destabilization of the myelin sheath.     

Size and Surface Charge Properties of Myelin Vesicles from Normal and Diseased (Multiple Sclerosis) Brain

Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross-sectional area of the vesicles was 5.69 +/- 0.17 micron 2 from normal myelin and 3.71 +/- 0.28 micron 2 for diseased myelin. Vesicle size was reduced to 4.08 +/- 0.21 micron 2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2+ during the preparation of the vesicles had no effect on the mean cross-sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 +/- 0.28 to 5.40 +/- 0.31 micron 2. Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was -5.169 +/- 0.193 X 10(-8) compared with -6.093 +/- 0.202 X 10(-8) m2 s-1 V-1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to -6.483 +/- 0.151 X 10(-8) m2 s-1 V-1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be -2.93 and -5.39 mV m-1, respectively. The phase transition temperature determined by wide-angle x-ray diffraction studies was 63 degrees C for normal myelin vesicles and 43 degrees C for MS myelin vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.