Characterization and protein analysis of myelin subfractions in rat brain: developmental and regional comparisons

Myelin preparations from the whole brains of 16-day old rats and from cortical regions and brainstem, respectively, of 40 day old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al. 1973). The total dry weight of myelin recovered from the 16 day old rats was only 2.4 mg/g fresh brain in comparison to 20 mg from adult brains. In 16 day old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16 day old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16 day old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40 day old rats was 4-fold higher than from cortical regions, and the percentage recovered in the ligt fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16 day old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin related membranes, although the heavy fraction from 16 day old rats also may be substantially contaminated with membranes which are unrelated to myelin.