Enzymic methylation of myelin basic protein in myelin

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.

Calcium- and cyclic AMP-regulated protein kinases of bovine central-nervous-system myelin

Bovine central-nervous-system myelin was found to contain both Ca2+-activated and cyclic AMP-dependent protein kinases. Each enzyme possesses unique solubility and substrate-specificity characteristics. The Ca2+-activated enzyme, like its substrate (basic protein), is probably deeply embedded in the neural membrane, whereas the cyclic AMP-dependent kinase appears to be much less tightly associated with myelin. Treatment of insoluble myelin fraction housing the Ca2+-activated kinase with phospholipase A2 and phospholipases A2 + C causes a decrease in its ability to become activated by Ca2+. This can be countered by phosphatidylserine and phosphatidylethanolamine. Whereas the activity of the Ca2+-activated membrane-associated kinase is inhibited by chlorpromazine, dibucaine, melittin and Triton X-100, it is activated by certain phorbol diesters (4 beta-phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate, 4 beta-phorbol 12,13-dibenzoate and 4 beta-phorbol 12,13-diacetate), which appear to exert this effect by lowering the concentration of Ca2+ normally required for the activation of this enzyme. Together these results suggest that the activation of the membrane-associated kinase by Ca2+ most probably requires certain lipids, perhaps those already present in the membrane.

Synthesis and incorporation of myelin polypeptides into CNS myelin.

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.

Antibody-independent complement activation by myelin via the classical complement pathway.

Murine or rabbit whole brain homogenates were shown to activate human complement via the classical pathway by an antibody-independent reaction. This activity required Ca++ ions. Anticomplementary activity in fractionated murine brain was found to reside in the myelin fraction and in purified myelin. It was absent, however, both from highly purified myelin basic protein (MBP) and from the MBP-free residue. Because purified MBP is a monomer and this protein exists in brain tissue largely as a dimer, the ability of the cross-linked form of MBP to activate complement was investigated. MBP, dimerized with difluorodinitrobenzene, was highly anticomplementary. The murine brain, inactive when taken from the newborn mouse, was shown to first acquire the capacity to activate complement at 7 d after birth. This finding is consistent with the report that the synthesis of myelin protein has been shown to be initiated in murine brain 8 d after birth. Complement activation by MBP could play an important role in the pathological changes observed in neurological disorders.

UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70-75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2-5% of the protein was recovered in the light-myelin fraction, and about 7-12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3-4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.