The activity of α-galactosyltransferase in cultured rat pheochromocytoma subcloned (PC12h) cells was examined using Gb3 as the acceptor for the galactose from UDP-galactose. The major reaction product was identified as galα1-3Gb3 based on its mobility on thin-layer chromatographic (TLC) plates and susceptibility to specific galactosidases. The enzyme activity in PC12h cells was the highest at pH 7.0 while the presence of Triton CF-54 (0.1%) and Mn2+ (5 mM) was required for its full activity. The apparent K(m) values for Gb3 and UDP-galactose were 57 and 17 μM, respectively. The enzyme activity in PC12h cells was compared with that in parent PC12 cells, in which galα1-3Gb3 is not expressed in an appreciable amount. In the enzyme reaction with exogenous Gb3, the enzyme activity in PC12h cells was about 1.5-fold higher than that in PC12 cells. In the absence of exogenous Gb3, this difference became even more pronounced; galα1-3Gb3 was generated from endogenous Gb3 at a much higher rate in PC12h cells than in PC12 cells. These findings suggest that the higher level of the α-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having galα1-3 terminal residues in the cells.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Lipid Research|
|State||Published - Jan 1 1992|
ASJC Scopus subject areas
- Cell Biology