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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.

A previous study of the selective solubility of myelin basic protein (MBP) of tissue extracts at pH 9.0 has raised issues of its quantitative recovery, and the differential solubility of its charge isomers. The pH-dependent solubility of proteins of acid extracts of delipidated tissue of bovine spinal cord was therefore reexamined. MBP of whole extracts was completely soluble up to pH 8.0 only, and less so by 25% at pH 9.0, and 43% at pH 10.0. The proteins other than MBP were virtually insoluble between pH 5.0 to 6.0, and 9.0 to 10.0. The solubility of the main charge isomers I to III of MBP of 18.5 kDa was found not to be affected by pH. Either pH 5.0 or 9.0 is therefore suitable for the selective isolation of MBP from whole tissue extracts, only pH 5.0 providing for the complete recovery of MBP. The pH-dependent solution behaviour was also examined following the separation of proteins of whole extracts by anion exchange chromatography at pH 10.4. Purified MBP and several related minor cationic components of lower molecular weight were soluble throughout. In contrast, the anionic proteins were only partly soluble between pH 4.0 to 10.0, i.e. by 4 to 20%. The results are consistent with specific protein-protein interactions of the proteins of whole extracts, either enhancing the solubility of non-MBP proteins, e.g. at pH 7.0, or impairing that of MBP between pH 8.0 to 10.0.

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.

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.

The three molecular forms of the anti-encephalitogenic protein, beta-SCP, gamma-SCP, and SCP-peptide were isolated in higher yield by a shortened procedure, which involved 1) extraction of bovine spinal cord (BSC) or bovine spinal roots (BSR) with 0.05 M sodium acetate buffer, pH 4.5, 2) batch absorption on CM-52 cellulose, 3) stepwise elution with sodium acetate buffers, pH 5.8, containing increasing concentrations of sodium chloride and finally, 4) removal of trace contaminants by gel-exclusion chromatography on Sephadex G-50 superfine. The m.w. of the purified proteins determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was 13,200 daltons. The same value for the molecular sizes was obtained by gel-exclusion chromatography by using 0.1% SDS in 0.05M sodium chloride as eluant. In the absence of SDS the molecular sizes estimated by gel exclusion chromatography ranged from 14,000 to 18,500. The amino acid compositions of the beta-SCP and gamma-SCP from BSC and BSR were similar except that beta-SCP from BSR lacked half-cystine whereas gamma-SCP from BSR contained three times as much half-cystine as the SCP forms prepared from BSC. All forms of SCP showed reactions of identity when compared by immunodiffusion analyses with a rabbit anti-bovine SCP serum; none formed precipitin lines with a rabbit anti-bovine myelin basic protein (MyBP) serum.

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.

Main encephalitogenic protein was isolated from bovine medulla spinalis and separated into homogenous polypeptide fractions by gel filtration; some of these fractions had the high encephalitogenic activity. The main protein and its fractions, possessing the encephalitogenic activity, caused a development of cross-reacting antibodies and a sensitization to intracutaneous administration of encephalitogenic preparations in animals. Non-encephalitogenic main protein sensitized animals only to this protein and did not induce the formation of distinct amount of antibodies. The amino acid composition of the encephalitogenic polypeptide is exhibited.

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)

Basic protein (SCP) known as antiencephalytogenic was isolated from the bull spinal cord and spinal roots. The protein is purified to a molecular homogeneous state, some of its physicochemical properties are studied. Molecular homogeneity of the protein is established by the method of disk electrophoresis in 15% polyacrylamide gel with sodium dodecyl sulphate present and by analytic ultracentrifugation. During electrophoresis in 15% polyacrylamide gel at pH 4.0 the protein movement occurs only in one zone, and at pH 7.0 three closely located zones are formed. The protein molecular weight determined by different methods is about 13500. The protein amino acid composition is determined and it is shown that 9.6 mole of amide nitrogen falls on 1 mole of the protein. The presence of the tertiary structure in the protein is supposed.

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.