An immunochemical investigation of ganglioside in rat brain myelin.

The chemical analysis of myelin isolated from brain tissue has indicated that gangliosides, and in particular the monosialoganglioside GMl, are constituents of this organelle (Suzuki etal., 1967, 1968; Suzuki, 1970; Ledeen etal., 1973). It is still possible, however, that these glycolipids may to some extent represent contamination of myelin fractions by oligodendroglial or axonal membranes. The finding that isolated particulate myelin is agglutinated by anti-ganglioside sera (Oxberry & Gregson, 1974) precludes axonal membrane as a major source of ganglioside, since such membranes would be trapped within the myelin particles and not be available for surface reactions. To examine further the possible heterogeneity of ganglioside distribution in rat brain myelin fractions these fractions have been further analysed by isopycnic centrifugation on linear sucrose density gradients. The absorption of anti-ganglioside antibodies by the resulting fractions followed closely the distribution of protein. The material did not produce a symmetrical distribution, heterogeneity being obvious at both the low and the high density edges. Electron microscopy demonstrated a preponderance of single membrane vesicles on the low-density edge and the presence of dense fibrillary structures at the high-density edge. The fractionated myelin was tested directly for complement fixation with both anti-ganglioside and anti-cerebroside sera. The distribution of activity was similar with both sera, but did not follow the protein distribution, being at a minimum where the protein was at a maximum. This supports the suggestion of heterogeneity within myelin fractions, but not with respect to a membrane that is particularly rich in ganglioside. It may be, however, that this is simply a reflexion of the degree of fragmentation of the myelin and the variation of surface area to protein content. This cannot be the complete explanation, however, because of the results on absorption of antibody. It is known that the complement-fixing activity of glycolipids is modified by their association with other lipids so the present results may indicate that both cerebroside and ganglioside are present in myelin fragments with differing combinations of other lipids. This may still be a reflexion of myelin fragmentation, with some particles retaining the original outer layers of the sheath and others, the small membrane vesicles perhaps, being derived from the deeper regions of the sheath. The reactivity of ganglioside at the surface of myelin particles was further investigated by the method of micro-electrophoresis. In 0.14~-NaCI and in the presence of normal rabbit serum, pH7.2, myelin particles had a mobility of 1.43+0.09(s.~)pm-' .s-' .V-'* cm-'; the mobility was lowered in the presence of anti-ganglioside serum to 1.2OkO.04 (s.D)pm-' .s-' ~V-'.cm-'.Thissupportsfurthertheconclusionthatgangliosideisexposed at the surface of the myelin particles. It also indicates that some proportion of the ganglioside is ionizable and contributes to the fixed charge of the particle surface. The decrease in mobility resulting from the interaction with anti-ganglioside serum corresponds to a decrease in surface-charge density of 805 e.s.u./cm2. Such a decrease in charge density is equivalent to a surface concentration of about 450pg of N-acetylneuraminic acid/g wet wt. of myelin. It has previously been shown that after the solubilization of myelin with lysophosphatidylcholine, and subsequent to centrifugation on a linear sucrose gradient, a discrete tertiary complex can be resolved exhibiting complement fixation with anti-cerebroside sera (Gregson et al., 1974). A similar investigation with anti-ganglioside sera indicates that the bulk of the ganglioside occurs as a complex equilibrating at 6-7.5 % sucrose, whereas the complement-fixing activity with anti-cerebroside serum equilibrates at 7.5-13 % sucrose. It is suggested that, although both cerebroside and ganglioside exist at the surface of myelin particles, after solubilization with lysophosphatidylcholine, these lipids may be distributed in different lipoprotein-lysophosphatidylcholine complexes, which may reflect their endogenous distribution.