The regulation of glycosphingolipid (GSL) synthesis in culture by fusion-competent (E63) myoblasts and fusion-defective (fu-1) cells was examined. Upon reaching confluency E63 cells fused to form multinucleated myotubes and demonstrated many characteristics of developing skeletal muscle including induction of creatine kinase activity and a shift in creatine kinase isozymes to the MM isoform. The fu-1 cells displayed none of these characteristics, despite the fact that both cells were cloned from the same parental myoblast line (rat L8). There was a transient increase in the synthesis of total neutral GSLs by E63 cells at the time of membrane fusion. In contrast, neutral GSL synthesis by fu-1 cells gradually decreased with time in culture. The major GSLs synthesized by both cell types were lactosylceramide and ganglioside GM3, with more complex structures being observed with prolonged time in culture. Several glycosyltransferase activities were assayed at varying times in culture. Generally, the changes in activities fell into three groups. One group was maximally activated at the end of the culture period (GalT-3, GalNAcT-1 and GalT-6). Another group was maximally activated during the time of active membrane fusion (GlcT and SAT-1). A third group was maximally activated at the time of cell contact and the beginning of membrane fusion (GlcNAcT-1 and GalT-2). In terms of the times of maximal activation there were few differences between E63 and fu-1 cells, with one notable exception. The activity of GalT-2 (lactosylceramide synthase) in E63 cells increased dramatically upon contact and the beginning of membrane fusion, whereas there were no changes in GalT-2 activity in fu-1 cells during time in culture. These results support our hypothesis that membrane glycosphingolipids play an important role in the differentiation of skeletal muscle cells.

A method is described whereby ganglioside GM1 can be quantitated directly on thin-layer chromatograms using cholera toxin subunit B conjugated to horseradish peroxidase and visualized with chloronaphthol. Overlay and color development were performed after separating gangliosides on nano-TLC plates, and fixing with polyisobutylmethacrylate. Absolute quantitation was realized using a Shimadzu CS-9000 integrating spectrodensitometer, scanning at 580 nm. A correlation coefficient of 0.98 was obtained in a linear range of detection from 10(-11) to 10(-16) moles. Statistical analysis revealed good reproducibility and over 99% of the added gangliosides remained with the chromatogram during all overlay and washing procedures. By comparison, standard chemical visualization by resorcinol-HCl was linear in the nanomole range with a detection limit of only 10(-10) moles. Since the carbohydrate portion of gangliosides immobilized in this manner is susceptible to the action of enzymes including neuraminidase, this technique can be applied to all structures of the gangliotetraose series.

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