Gel filtration of myelin using 2,2,2-trichloroethanol as the solvent.

The use of 2,2,2-trichloroethanol as a solvent for myelin from both the central and peripheral nervous systems is described. Concentrated, optically clear solutions of lyophilized myelin in this solvent are stable for weeks. The preparation of highly concentrated myelin proteins by gel filtration in trichloroethanol is described.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2',3'-cyclic nucleotide 3'-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G(M1). A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2′,3′-cyclic nucleotide 3′-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G_M1. A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Lipid and basic protein interaction in myelin

1. Purified myelin labelled with [(3)H]myo-inositol or [1-(14)C]acetate was incubated with trypsin or acetylated trypsin at 37 degrees C, pH8.0 for 30min. 2. After incubation and centrifugation analysis of the myelin pellet showed marked digestion of basic protein on polyacrylamide-gel electrophoresis. Proteolipid and Wolfgram proteins remained unchanged. 3. A loss of 15% of total protein and loss of all classes of lipids was also found. Most significant lipid losses were phosphoinositides, phosphatidylserine and sulphatide. 4. A low-density material containing more phospholipid than cholesterol and galactolipid was isolated from the supernatant obtained after centrifugation of trypsin-treated myelin. 5. Interaction of sulphatide and myelin basic protein was shown to take place in a biphasic system. Basic protein does not form any complex either with cerebroside or cholesterol in the same solvent system. 6. The release of acidic lipids from myelin suggests that they may be linked to basic protein by ionic forces and the neutral lipids may be by lipid-lipid interactions. 7. The relevance of these studies as a model of brain degeneration is discussed.     

The identity of a myelin-like fraction isolated from developing brain

1. A myelin-like membrane fraction was isolated from developing rat brain by a new method. 2. The chemical composition and morphology of the fraction are described. 3. The myelin-like fraction is similar to myelin in characteristic enzyme activity but differs in the absence of basic protein and cerebrosides. No similarity to other subcellular fractions was observed. 4. It is suggested that the myelin-like fraction is a stage in the formation of compact myelin from glial plasma membrane. 5. ;Early' myelin consists of the myelin-like and compact myelin fractions from developing brain.     

Large Scale Preparation of Myelin Basic Protein from Central Nervous Tissue of Several Mammalian Species

The wide-spread use of and demand for myelin basic protein for immunologic studies has prompted us to re-examine the details of its isolation from CNS tissue of various species. The procedure described in this communication for the isolation and purification of myelin basic protein does not require column chromatography and is therefore suitable for large scale preparation of a reasonably pure product with simple laboratory equipment. If certain precautions are taken, the yield and quality of the product are reproducible. Certain contaminants which may accompany myelin basic protein during purification by procedures currently in use are pointed out, and their possible influence on the immunologic behavior of myelin basic protein is discussed. Suitable electrophoretic techniques for the detection of these contaminants as well as details for their removal from the myelin basic protein are described.     

Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.     

Meylin types with different protein components in the same species

Abstract— Myelin was isolated from bovine optic nerve, cerebral white matter, spinal cord white matter and peripheral nerve (intradural spinal roots). The freeze-dried myelin completely dissolved in phenol-formic acid-water (14:3:3, w/v/v), and acrylamide gel electrophoresis of the myelin proteins was performed with this solvent. Qualitative and quantitative differences were observed in the myelin proteins from the various regions of the CNS. Myelin of peripheral nerve contained proteins that are apparently unique to it and which are not found in the myelin of the CNS.     

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis.

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca2+. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.     

Polyacrylamide gel electrophoresis of myelin proteins: a caution.

Some variables involved in the preparation of rat brain myelin proteins for polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate were studied. Under mild conditions of solubilization the resultant gel patterns were relatively insensitive to the β-mercaptoethanol (ME) concentration in the protein solvent used for solubilization of myelin proteins. However, if the samples were boiled in the presence of ME (a standard procedure for disruption of metastable aggregates of membrane proteins), a major myelin protein, proteolipid protein, as well as some minor proteins were preferentially excluded from the gel. This effect was proportional to the ME concentration.     

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca²⁺. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.     

Phosphorylation of myelin proteins: Recent advances

Since it was first described 25 years ago, phosphorylation has come to be recognized as a widespread and dynamic post-translational modification of myelin protein. In this review, the phosphorylation characteristics of myelin basic protein, protein zero (P₀), myelin-associated glycoprotein and 2′3′ cyclic nucleotide 3′-phosphodiesterase are summarized. Emphasis is placed on recent advances in our knowledge concerning the protein kinases involved and the sites, of phosphorylation in the amino acid sequences, where known. The possible roles of myelin protein phosphorylation in modulating myelin structure, the process of myelin assembly and mediation of signal transduction events are discussed. Special issue dedicated to Dr. Marion E. Smith.     

Determination of myelin marker enzymes in the blood serum in peripheral nervous system diseases

The activity of 2',3'-cyclonucleotide-3-phosphohydrolase (CNP) and leucine aminopeptidase (LAP) was studied for the first time in the blood of patients with diseases of the peripheral nervous system. A spectrophotometric method for CNP activity detection was described. The activity absent from the blood of normal subjects was detectable with statistical significance in the patients with the diseases in question. Analogous results were obtained in the animals with an experimental injury to the myelin coat of the peripheral nerves. Study into the activity of LAP in the pathological conditions under consideration revealed no significant deviations. It is emphasized that CNP may transfer to the blood serum from myelin decay foci and that the method described may be used for diagnostic purposes.     

The composition of the myelin proteins of the peripheral nervous system

Abstract— A method for the isolation of peripheral nerve myelin is described. Peripheral nerve myelin differs from centrum ovale myelin in its amino acid composition, in that it contains a greater proportion of protein that is digestible with trypsin and pepsin, and in the insolubility of its proteins in chloroform–methanol.     

A freeze-etch study of the central myelin

The intramembranous organization of the interparanodal myelin lamellae in CNS was investigated by means of the freeze-etching technique. The density of the IMPs is about equal on both P- and E- fracture faces of every myelin sheath, but varies between the myelin sheaths. The morphology of the myelin tight junctions is also described. The nature of the IMPs and the functional significance of their mode of distribution are discussed.     

Synthesis of adenosine triphosphate by myelin of spinal nerves of rabbit

Abstract—

• 1 The myelin fraction isolated by isopycnic gradient centrifugation from rabbit nerve is able to synthesize ATP at substrate level through the Embden-Meyerhof pathway. Suitable conditions are described to preserve the association of glycolytic enzymes with isolated myelin.
• 2 Except for phosphofructokinase and ketose-1-phosphate aldolase, all the remaining glycolytic enzymes are present in the myelin. A wide divergence was found in the firmness of the association of individual glycolytic enzymes with myelin under the condition of isolation; some, like glucosephosphate isomerase and glyceraldehydephosphate dehydrogenase were retained in high percentage (about 60 per cent of the activity of the homogenate is myelin-bound); others were weakly bound (no more than 7–6 percent of the lactate dehydrogenase activity of the homogenate is myelin-bound).
• 3 By using glyceraldehyde-3-phosphate as substrate for glycolysis, about 25 per cent of the total glycolytic activity of rabbit-nerve homogenate is associated with the myelin.
• 4 Glucosephosphate isomerase and lactate dehydrogenase may be extracted from and readily recombined with the myelin.

     

Preparation and properties of monoclonal antibodies to myelin basic protein and its peptides

Techniques are described which have enabled the production and characterisation of monoclonal antibodies to myelin basic protein. These are shown by enzyme immunoassay to react with six different epitopes. Two of these are to peptide 82–91, a region claimed to be present in the spinal fluid of patients with demyelinating disease. One of these, an IgG_2a, is shown to react only with peptides in which the 91–92 phe-phe bond has broken. The other, an IgM, also reacts with whole myelin basic protein. The IgG_2a antibody is shown to have an affinity suitable for use in immunoassay of peptide 82–91. The enzyme immunoassay procedures described help to minimise the work load involved in the preparation and characterisation of monoclonal antibodies to this protein.     

Role of lipid interactions in autoimmune demyelination

A morphological transformation involving loss of adhesion between myelin lamellae and formation of myelin vesicles has been described as a mechanism for demyelination in multiple sclerosis and marmoset experimental allergic encephalomyelitis (EAE). Although protein interactions are involved in maintaining normal myelin structure, we describe here how lipids contribute to myelin stability and how lipid changes in EAE, including increases in lipid polyunsaturation and negatively charged phosphatidylserine (PS), promote demyelination. Three physico-chemical techniques were used to identify these changes: (1) Langmuir monolayer isotherms indicated that EAE white matter lipids were significantly more "expanded" (fluid) than controls. (2) NMR spectroscopy indicated that EAE myelin lipids were more polyunsaturated than controls. (3) High-performance liquid chromatography (HPLC) with an evaporative light scattering detector indicated increased PS in EAE compared to controls, while sphingomyelin (SM), sulfatides and phosphatidylcholine (PC) were decreased. We present a physical model considering electrostatic, van der Waals and undulation forces to quantify the effect of these changes on myelin adhesion at the extracellular interface. Taken together, the isotherm, NMR, HPLC and modeling results support a mechanism for autoimmune demyelination whereby the composition of myelin lipids is altered in a manner that increases myelin fluidity, decreases myelin adhesion, increases membrane curvature, and promotes vesiculation.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN : II. Optic Nerve

The dehydration of rat optic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. A correlation is established between electron micrographs of the myelin sheath and corresponding small-angle x-ray diffraction patterns. The modifications of the optic nerve myelin layers during drying were very similar to those described in more detail for the myelin of frog sciatic nerve. The most striking difference was that the system of fine layers characteristic of the fully dried myelin was much more extensive in the case of the optic nerve, and the layer thickness was significantly greater than the corresponding layer in the frog sciatic nerve preparation. The significance of these correlations is discussed.     

Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol: acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.