Comparison of N-acetylglucosaminyltransferase V activities in Rous sarcoma-transformed baby hamster kidney (RS-BHK) and BHK cells.
Recent studies have demonstrated that Rous sarcoma virus-transformed baby hamster kidney (RS-BHK) cells express twofold higher levels of those N-linked oligosaccharides that contain the sequence [GlcNAc-beta(1,6)Man (1,6)] compared to nontransformed parental BHK cells (Pierce and Arango, J. Biol.Chem. 261, 10772 [1986]). We have investigated in RS-BHK and BHK cells the activity of UDP-GlcNAc:alpha-D-mannoside beta(1,6)N-acetylglucosaminyltransferase V, the enzyme that begins the synthesis of the sequence that is increased in the RS-BHK cells. We have measured GnT V activity using UDP-[3H]-GlcNAc and a synthetic oligosaccharide acceptor, GlcNAc beta(1,2)Man alpha(1,6)Man beta-O-(Ch2)8COOCH3, separating the radioactive product by a newly devised reverse-phase chromatographic technique. Assayed under optimal conditions, the specific activity of GnT V is about fourfold higher in RS-BHK sonicates than in BHK sonicates, suggesting that this increase in activity may be the primary mechanism that causes the increase in [GlcNAc beta(1,6)Man] sequences in the RS-BHK cells. The apparent Km values of the enzymes in RS-BHK and BHK cell sonicates for UDP-GlcNAc and the synthetic acceptor are similar, as are the pH optima. These results suggest that the increase in GnT V-specific activity in RS-BHK cells is not caused by the presence in these cells of a GnT V with markedly different kinetic properties.
Arango
J
J
Department of Anatomy and Cell Biology, University of Miami School of Medicine, FL 33101.
Pierce
M
M
D000117:Acetylglucosamine / Q000032:analysis; D000818:Animals; D001358:Avian Sarcoma Viruses; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D006224:Cricetinae; D005964:Glucosyltransferases / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases; D011120:Polyomavirus; D014530:Uridine Diphosphate / Q000032:analysis; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D014537:Uridine Diphosphate N-Acetylglucosamine; D014530:Uridine Diphosphate; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine