Activity of 3-ketosphinganine synthase during differentiation and aging of neuronal cells in culture

Changes in the enzyme 3-ketosphinganine synthase activity in rat cerebellar granule cells in culture were studied during differentiation and aging. The enzyme activity with palmitoyl-CoA and stearoyl-CoA, precursors of, respectively, C18-sphinganine and C20-sphinganine, was studied on the total cell homogenate using radioactive serine. The enzyme assay was performed by thin-layer chromatography (TLC) separation of the enzyme reaction mixture, and the resultant radioactive 3-ketosphinganine was identified by chromatographic comparison with a chemically synthesized 3-ketosphinganine, and quantified by determination of the TLC radioactivity distribution on the basis of the radioactivity content of cell lipid extract that was determined by scintillation counting. Using palmitoyl-CoA, the enzyme activity progressively increased from 40 to 54 pmol of 3-ketosphinganine/mg cell DNA per min in the first 8 days and then progressively decreased, and was 39 pmol of C18-(3-ketosphinganine)/mg cell DNA per mill at day 22 in culture. For stearoyl-Coh the enzyme activity was very low at day one and then increased to a constant value of about 15 pmol of C20-(3-ketosphinganine) /mg cell DNA per min. These results are in good agreement with the finding that the ganglioside species that contain C18-sphingosine increase during cell differentiation and remain constant during cell aging, while the ganglioside species that contain C20-sphingosine continuously increase during both cell differentiation and aging.