Abstract
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.
Highlights
IThese findings suggest that the higher level of the a-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having galal-3 terminal residues in the cells.-Pal, S., M
Glycosphingolipids are important constituentsof the plasma membranaend constitupteart of the glycocalyx network of the cell surface. They have been implicated to participate in diverse cellular functions: receptorsforhormones, virus, and bacteria, cell-cell interactions,proliferation, and differentiation [1, 2]
We foundthatuniqueneutral glycosphingolipids having galal-3 terminal residues, including galal-3Gb3, accumulated in a subclone of cultured rat pheochromocytom(Pa C12h) cells [3, 4]. These glycolipids are not expressed in appreciable amounts intheparent PC12 cells ( 3 )
Summary
IThese findings suggest that the higher level of the a-galactosyltransferase activity in PC12h cells may, at least in part, be responsible for the accumulation of unique neutral glycosphingolipids having galal-3 terminal residues in the cells.-Pal, S., M. We foundthatuniqueneutral glycosphingolipids having galal-3 terminal residues, including galal-3Gb3, accumulated in a subclone of cultured rat pheochromocytom(Pa C12h) cells [3, 4] These glycolipids are not expressed in appreciable amounts intheparent PC12 cells ( 3 ). To examine the functional role of these glycosphingolipids, it would be important to clarify the regulatory mechanism fortheir metabolism in PC12hcells In this investigation, we characterized the activity of UDP-. Galactose:Gb3 a-galactosyltransferase in PCl2h cells, and demonstrated that the higher level of the enzyme activity in PC12hcells might,at least partially, be responsible fortheaccumulation of these unique glycosphingolipids in the cells
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