Subcellular Fractionation of Rat Sciatic Nerve and Specific Localization of Ganglioside LM1in Rat Nerve Myelin

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Abstract
Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctasylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (< 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were –70–75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (–40%) and GM1 (–20%). LM1 in these fractions was minimal (less than –5%). Significant amounts of GM3 with N-glycolylneuraminic acid (–10%) and GM1b (4–14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.