Notes: Abstract— Partially purified myelin from brains of 17-day-old rats was separated into 4 subfractions on a discontinuous sucrose gradient by virtue of heterogeneity in density and particle size. The protein composition of each subfraction was determined by densitometry following separation of proteins on polyacrylamide gels in buffers containing sodium dodecyl sulphate. The major proteins studied included two basic proteins, proteolipid protein, the major high molecular weight protein (W) and a group of high molecular weight proteins.The percentage of high molecular weight proteins decreased sequentially from fraction D to A, that of the W protein remained constant, while relative amounts of the two basic proteins increased. Proteolipid protein concentration also increased as a percentage of the total protein from fraction D to B, but the uppermost fraction. A, had a markedly lower amount than fraction B. At 1 h after intracranial injection of [3H]leucine, the specific radioactivity of the basic and proteolipid proteins decreased from fraction D to B, with proteolipid protein in fraction A again anomalous (specific radioactivity higher than expected). These results are consistent with (but do not prove) a precursor-product relationship for individual proteins from denser to lighter subfractions, with the exception of myelin subfraction A.Experiments involving time staggered injections of a [14C] and later a [3H] labelled amino acid gave data which demonstrated that the W and basic proteins were added simultaneously (or with delays of much less than 20 min) to all of the subfractions, while proteolipid protein was added sequentially, from lower to upper fractions on the gradient. This double isotope technique also confirmed our previous observations that proteolipid protein shows a lag in entry into myelin compared to basic protein.

Notes: Abstract— Partially purified myelin from the brains of 17-day-old rats was separated into 4 subfractions on a three-step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor-product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2-3H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [3H]galactose was the precursor. These data are consistent with (but do not prove) a precursor-product relationship for individual lipids from the densest to the lightest subfraction.Another experimental design involving time staggered injections of [3H] and [14C] precursors was developed which enables a more definitive result with regard to precursor-product relationships to be obtained. A precursor-product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor-product relationship. Ihe isotope ratio should be constant. Such experiments were done with [3H] and [14C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [3H] and [14C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.

Notes: Abstract— Seventeen-day-old rats were injected intracranially with [3H]leucine, then sacrificed between 1 and 24 h. Myelin was prepared from the brains on discontinuous sucrose gradients and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulphate. Proteins were stained with acid Fast Green and the distribution was quantitated by densitometry. The gels were then sliced and the radioactivity in each slice was determined. Between 1 and 24 h, the radioactivity in proteolipid protein increased from 18% to 37% of the total radioactivity in the proteins of isolated myelin. During this same period, the per cent distribution of radioactivity in basic and Wolfgram proteins remained constant while that in the remaining high molecular weight proteins decreased. Similar results were also obtained with [3H]glycine as a precursor. The relative specific activity of all of the myelin proteins increased between 1 and 6 h, then remained constant between 6 and 24 h. At 1 h, proteolipid protein reached only 25% of its maximal (6 h) relative specific radioactivity, while the other two proteins reached 50% of maximum. These results indicate a lag in the appearance of labelled amino acids in proteolipid protein relative to the other myelin proteins.