Accumulation of [3H]sialyl-conjugates in sialidosis (sialidase-deficient) fibroblasts cultured in the presence of [3H]-N-acetylmannosamine.
Skin fibroblasts from normal individuals and a patient with the infantile form of sialidosis were cultured for up to 72 h in medium containing [3H]-N-acetylmannosamine. The sialidosis fibroblasts consistently accumulated more labeled compound(s) than the control cells, i.e. 37-88% more cpm per mg protein. Precipitation of sonicates of these cells with 10% trichloracetic acid, TCA, demonstrated that the excess radioactivity in the sialidosis fibroblasts was in one or more TCA soluble compounds. There was no detectable difference in the amount of label in the TCA insoluble material. The TCA soluble, labeled, material from the sialidosis and the control fibroblasts was separated, isolated and purified on AG1-X8, QAE Sephadex A-25 and Bio-Gel P-4 chromatography columns. Analysis of the isolated material showed the excess radioactivity in the sialidosis fibroblasts to be due to increased levels of [3H]sialic acid covalently bound to a variety of anionic sialyl conjugates. These compounds have been separated and partially purified. Finally, acid hydrolysis and chromatographic analysis of the TCA insoluble fractions showed that greater than 80% of the label in this material was also due to [3H]sialic acid. There was no detectable difference between the control and the sialidosis patient in the amount of label in this fraction.
D002239:Carbohydrate Metabolism, Inborn Errors / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D006595:Hexosamines / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009439:Neuraminidase / Q000172:deficiency*; D009844:Oligosaccharides / Q000096:biosynthesis / Q000302:isolation & purification; D012016:Reference Values; D012794:Sialic Acids / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*; D014316:Tritium
D006595:Hexosamines; D009844:Oligosaccharides; D012794:Sialic Acids; C016735:sialooligosaccharides; D014316:Tritium; D009439:Neuraminidase; C002022:N-acetylmannosamine