Journal of Lipid Research, Vol 33, 411-417, Copyright © 1992 by Lipid Research, Inc.
UDP-galactose:globotriaosylceramide alpha-galactosyltransferase activity in rat pheochromocytoma (PC12h) cells
S Pal, M Saito, T Ariga and RK Yu
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
The activity of alpha-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 gal alpha 1-3Gb3 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 Km values for Gb3 and UDP- galactose were 57 and 17
microM, respectively. The enzyme activity in PC12h cells was compared with
that in parent PC12 cells, in which gal alpha 1-3Gb3 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; gal alpha 1- 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 alpha-galactosyltransferase activity in PC12h cells
may, at least in part, be responsible for the accumulation of unique
neutral glycosphingolipids having gal alpha 1-3 terminal residues in the
cells.
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