UDP-galactose:globotriaosylceramide alpha-galactosyltransferase activity in rat pheochromocytoma (PC12h) cells.
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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Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Saito
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M
Ariga
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Yu
R K
RK
D000310:Adrenal Gland Neoplasms / Q000737:chemistry / Q000201:enzymology*; D000818:Animals; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005700:Galactosyltransferases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009368:Neoplasm Transplantation; D010673:Pheochromocytoma / Q000737:chemistry / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D003902:Detergents; D008670:Metals; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase