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The concurrent release of myelin basic protein (MBP) and extrinsic proteinases from isolated myelin membranes by aqueous solvents of high ionic strength is considered circumstantial evidence of a presumptive mutual interaction in situ. The joint release of proteins and proteinases from myelin membranes of bovine brain, depending on the ionic strength of aqueous solvents, was therefore examined; 25 mM Tris buffer released an average 1.4% of total myelin protein. It was attributable to about 25 different electrophoretic bands, but no apparent MBP. However, the extract potently mediated the limited proteolysis of added MBP at pH 4.0, 5.6, and 9.0. Because of the pH and the effects of specific inhibitors, proteolysis appears to be owing to activities of cathepsin B and D, and an alkaline metalloproteinase. The subsequent extraction of myelin membranes with buffered 300 mM NaCl released an additional 20% of total myelin protein, mainly MBP. The extracts, unlike those of untreated myelin membranes, no longer cleaved MBP at pH 5.6 and 9.0, and did so only slightly at pH 4.0. The results indicate that the bulk of soluble myelin-associated proteinases is much less tightly bound than MBP. The weak binding of the former and the prevalence of lysosomal cathepsin B- and D-like activities suggest that during their isolation, myelin membranes may adsorb soluble cellular proteins of tissue homogenates. At any rate the washing of myelin membranes with dilute buffer was found to largely remove soluble proteinase activities that are otherwise associated with salt-soluble MBP of myelin.

To study the role of HIV-1 gp120 in loss of myelin in HIV encephalopathy, the binding of gp120 to various types of neural cells and its effects on myelination were examined in rat primary brain culture. Double-staining of cultured cells with gp120 and specific antibodies for different neural cell types showed that gp120 bound to most of the galactocerebroside (GalC)-positive oligodendrocytes, a small population of type-2-like astrocytes and a few small neurons. Gp120 did not bind to type-1-like astrocytes, most neurons, or to macrophage/microglia. To assay myelination, cells were bathed in a myelination medium containing chick embryo extract and high glucose, with or without gp120. Seven days after the application, myelination in the culture was observed morphologically and by staining with anti-myelin basic protein (MBP) antibody, and was found to be significantly inhibited by the addition of gp120 (50-100 nM). The processes of oligodendrocytes were reduced in length and arborization relative to the control, but MBP production by oligodendrocytes was unaffected. These results show that gp120 can cause a functional disorder of oligodendrocytes and thus could underlie the diffuse loss of myelin sheaths of HIV encephalopathy.

Sodium chloride extracts obtained from purified bovine brain myelin were found to contain proteolytic activity capable of degrading isolated myelin basic protein as assessed by SDS gel electrophoresis. Using gels copolymerized with gelatin as substrate, two bands at about 54 and 117-125 KDa, respectively, were detected. Activity corresponding to the 54 KDa band was inhibited by zinc. Data presented in this article suggest that proteolytic activity can be released from the myelin sheath in water-soluble form and recognize MBP as substrate.

Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition.

Myelin basic protein (MBP) was purified from guinea pig spinal-cord in a native-like form retaining the binding to all the myelin lipids. Since the guinea pig MBP was found to be much more labile than the corresponding MBP from bovine brain, the original procedure based on the use of hydroxyapatite and detergents was slightly modified as reported here. The product of this purification, lipid-bound MBP, may represent an alternative to lipid-free MBP for the induction, the study and the treatment of experimental allergic encephalomyelitis.

Human and rat myelin preparations were incubated with varying concentrations of trypsin and plasmin to determine the effects of these proteolytic enzymes on myelin-associated glycoprotein (MAG), basic protein, and other myelin proteins and to compare the effects with those of the neutral protease that was reported to be endogenous in myelin. Basic protein was most susceptible to degradation by both trypsin and plasmin, whereas MAG was relatively resistant to their actions. Under the assay conditions used, the highest concentrations of trypsin and plasmin degraded greater than 80% of the basic protein but less than 30% of the MAG, and lower concentrations caused significant loss of basic protein without appreciably affecting MAG. Neither trypsin nor plasmin caused a specific cleavage of MAG to a derivative of MAG (dMAG) in a manner analogous to the endogenous neutral protease. Thus the endogenous protease appears unique in converting human MAG to dMAG much more rapidly than it degrades basic protein. MAG is slowly degraded along with other proteins when myelin is treated with trypsin or plasmin, but it is less susceptible to their action than is basic protein.

A purified myelin fraction was prepared from myelin deficient (mld) mutant mice and normal littermates (20 to 25 days old). Mld brains contained only 5% of the amount of myelin present in controls. Myelin isolated from mld brains had a normal lipid and protein content. The lipid composition of mld myelin was also normal. Myelin basic protein (MBP) was nearly missing in mld myelin. At the electron microscope, in mld myelin, the major dense line was often missing and the myelin lamellae were uncompacted. The high specific radioactivity of the myelin associated glycoprotein and sulfatides, two myelin constituents which are not affected by the mutation, indicate a high turnover rate of myelin membranes in the mld mutant. Furthermore, the presence of increased amounts of cholesterol esters in mld brains suggests that myelin in mld mutants is unstable. We propose that MBP, which is responsible in the central nervous system for myelin compaction, is responsible for the stability of this membrane.

Cation-dependent acid protease activity associated with isolated human myelin was inhibited by pepstatin A, or enzymatic reactions were suppressed altogether by maintaining samples at 0 degrees C rather than 37 degrees C to examine whether or not they are involved in the extraction of myelin basic protein (MBP) by increased ionic strength. These measures largely abolished the degradation of MBP by acid protease activity associated with myelin, whereas the extraction of protein was only slightly diminished. Electrophoresis revealed that soluble protein was exclusively accounted for by undegraded MBP. Acid proteolysis, therefore, appears not to be involved in the cation-mediated removal of MBP from myelin. It is suggested that this mechanism may account for the appearance of undegraded MBP in body fluids, as well as for its pathologically increased degradation once it has become soluble.

Incubation of bovine CNS myelin with phospholipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholipase C from Clostridium perfringens, phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9-12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.

Myelin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5'-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2',3'-cyclic nucleotide-3'-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiverers, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hypothetically, the 5'-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.

When lyophilized myelin prepared from canine brain white matter was extracted with a solvent mixture of n-propanol-n-hexane (2:3, v/v), it was found that the extract contained less amounts of sulfatide and phosphatidylserine and no protein like so-called proteolipid-protein in comparison with the total chloroform-methanol (2:1, v/v) extract of myelin. Thus, the propanol-hexane myelin residue was re-extracted with chloroform-methanol (2:1, v/v) and the extract was subjected to UV-spectroscopy, TLC and slab SDS-gel electrophoresis, respectively. The UV-absorption pattern of the extract showed a maximum band due to protein at about 280 nm. The lipid portion was found to be predominantly sulfatide (63.6%), phosphatidylserine (26.2%) and other lipids (10.2%), while the major portion of protein was basic protein. The amino acid composition of the extract was also very similar to that of a control porcine basic protein. The major normal fatty acids of sulfatide were C24:0 (34.5%), C24:1 (13.7%), C23:0 (8.3%) and C18:0 (16.0%), while the hydroxy acids were predominantly C24:0 (44.1%), C24:1 (22.2%) and C23:0 (10.4%). Also, the fatty acids of phosphatidylserine were mainly C18:0 and C18:1. The solubilization of the basic protein-acidic lipid complex (about 2.56 by weight in the ratio of protein to lipid) in chloroform-methanol suggested the interaction of the basic protein with acidic lipids which might be related to the molecular organization in myelin. The basic protein-acidic lipid complex was also found to have an encephalitogenic activity which seemed to be a little different from that of basic protein itself.

Hartley guinea pig central nervous system (CNS) myelin has been purified and fractionated into its protein and lipid components. Experimental allergic encephalomyelitis (EAE) was induced in juvenile strain 13 guinea pigs with both lyophilized and fresh 'wet' myelin. However, a larger dose of lyophilized myelin was required to induce chronic EAE. Total myelin lipids, galactocerebrosides, gangliosides, phospholipids or proteolipids were combined with a non-encephalitogenic dose of myelin basic protein (MBP) and injected in juvenile Hartley guinea pigs. No clinical or histological manifestations of disease were observed. Parameters of immune functions indicated that the total myelin lipids augmented cell-mediated immune responses as measured by in vitro lymphocyte transformation and by a significant decrease in the percentage of peripheral early T cells. Only the proteolipids elicited delayed hypersensitivity reactions. Animals that received the phospholipid-MBP combination showed no changes when compared to animals injected with MBP alone. The results suggest that although the myelin lipids did not act synergistically with a non-encephalitogenic dose of MBP to induce EAE, they induced immunological changes and potentiated the immune response to MBP.

Encephalitogenic protein fraction (BEC) was isolated from bovine brain tissue by extraction with salt-ethanol mixture at neutral pH, instead of employing dilute mineral acid. The fraction BEC was separated into two fractions. An acid-soluble protein was encephalitogenic and the major component was very alike to the basic protein of myelin (Al). The other was acid-insoluble acidic protein that was not encephalitogenic even at a dose of 100 mug. The acidic protein formed an insoluble complex with Al rotein which was purified by Eylar's method. Encephalitogenic activity of the complex was higher than Al protein in young guinea pigs when injected with complete Freund's adjuvant.However, this enhancement of encephalitogenic activity was not observed in aged guinea pigs. The complex showed higher blastogenic activity than Al protein alone with peripheral blood lymphocytes from guinea pigs immunized with Al protein and complete Freund's adjuvant. These results show that an adjuvant-like acidic protein is present in brain tissue and the complex with Al protein enhances the induction of experimental allergic encephalomyelitis (EAE).

A procedure for large scale isolation of myelin basic protein (BP) has been modified to insure BP preparations free of neutral proteinase activity. Fractions were monitored by electrophoretic analysis of BP solutions incubated under various conditions of temperature and pH. Maximum degradation of human BP prepared by the old batch procedure occurs at pH 7, approximately 47 degrees C. BP preparations obtained by the new procedure, as well as BP preparations purified by CM-cellulose chromatography, are stable under these conditions. The latter, however, do undergo significant breakdown at pH 9, 100 degrees C. The results suggest that the degradation observed under these conditions is non-enzymatic in nature.

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable alpha-helix, beta-structure, beta-turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high beta-sheet content and virtual absence of alpha-helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the beta-structure over the alpha-helical conformation. In order to define those beta-sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted beta-strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential beta-strands in the predicted secondary structure were then ordered into beta-sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a "Greek key" beta-barrel, consisting of eight antiparallel beta-strands with a two-stranded ribbon of antiparallel beta-structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative beta-strand orderings yield three specific models having different interstrand connections across the barrel ends.

Isolation of several of the charge isomers from both normal and multiple sclerosis (MS) myelin basic protein (MBP) was achieved on CM-52 columns at pH 10.6. In liquid x-ray diffraction and aggregation experiments, corresponding charge isomers were equally effective in the formation of multilayers, demonstrating the dominant role of overall net positive charge. These studies demonstrated that the change in overall charge of MBP of one net positive charge was sufficient to produce large changes in aggregation and in multilayer formation. The x-ray diffraction experiments showed that component 1 was twice as effective as component 2 although they differed in charge by a single positive charge. Component 3 was less effective than component 2 and component "8" was not effective at all. Vesicle aggregation also showed a dependence on net positive charge. In order of decreasing effectiveness, component 1 greater than component 2 greater than component 3 greater than component "8". Since overall charge on MBP is determined by contributions from the various charge isomers, the relative proportions of these charge isomers favoring the less cationic components could explain the observation that MBP from MS victims was less effective than MBP from normal brain in vesicle aggregation and multilayer formation. The isolation of myelin-containing white matter fractions from both normal and MS tissue in which the loss of some of the most cationic charge isomers was correlated with presence of less compact myelin supports this hypothesis.

Myelin was purified from the spinal cords of normal mice and mice heterozygous for the shiverer mutation, and measurements were made of the major myelin proteins and lipids and the specific activities of three myelin-associated enzymes. The myelin purified from the spinal cords of the heterozygotes (shi/+) was deficient by 30-40% in yield and had an apparently unique composition. In particular, when compared with normal mouse spinal cord myelin, there were more high-molecular-weight protein, less myelin basic protein, a higher protein-to-lipid ratio, and higher specific activities of 2',3'-cyclic nucleotide-3'-phosphohydrolase (EC 3.1.4.37) and carbonic anhydrase (EC 4.2.1.1) in the myelin purified from the shi/+ animals. These abnormalities were reflected in the composition of shi/+ whole spinal cord, where the protein-to-lipid ratio was intermediate between the respective values for +/+ and shi/shi spinal cords. Whole brains from shi/+ mice showed deficiencies in galactocerebroside and galactocerebroside sulfate and an increase in total phospholipid, and the lipid composition in the brains of the shi/shi mice was similar to that reported for another dysmyelinating mutant, quaking. The findings provide the first values for the lipids in normal mouse spinal cord myelin and show that heterozygotes are affected by the shiverer mutation. The observations imply that there can be considerable deviation from the normal CNS myelin content and composition without apparent qualitative morphological abnormalities or loss of function and that the amount of myelin basic protein available during myelination may influence the incorporation of other constituents into the myelin membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental allergic neuritis has been produced in the inbred Lewis rat in the absence of experimental allergic encephalomyelitis (EAE) using bovine intradural root myelin. The lack of EAE is probably because P1 is only weakly encephalitogenic in the rat. One of the basic proteins of bovine peripheral myelin, P2, was isolated and demonstrated to be pure by amino acid analysis and SDS PAGE. It was found to have a molecular weight of 15,400 and contained 4 mol 1/2-cystine/mol. This P2 was found to be highly neuritogenic and is probably the sole neuritogenic antigen in this system. The successful demonstration of its neuritogenicity must be due in large part to the use of the inbred Lewis rat and bovine P2, but an explanation could also involve the omission of denaturing organic solvents, the prevention of oxidative denaturation and presumably the fact that any changes which may occur are not sufficient to prevent recognition of the active site by the immune system of the inbred Lewis rat. P2 was neuritogenic down to 5 micrograms/animal. Its activity was enhanced by but not dependent on the presence of Mycobacterium in the adjuvant. This suggested that release of P2 could possibly break tolerance and produce an auto-immune disease such as the Guillain--Barre syndrome.