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 min at day 22 in culture. For stearoyl-CoA 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.
Chigorno
V
V
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
Negroni
E
E
Nicolini
M
M
Sonnino
S
S
D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D019735:Carbon-Carbon Ligases; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D008025:Ligases / Q000032:analysis / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009474:Neurons / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D009994:Osmolar Concentration; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D000214:Acyl Coenzyme A; D011506:Proteins; C002882:ketodihydrosphingosine; D010171:Palmitoyl Coenzyme A; C017725:stearoyl-coenzyme A; D012694:Serine; D008025:Ligases; C023945:3-ketosphinganine synthetase; D019735:Carbon-Carbon Ligases; D013110:Sphingosine