Comparison of N-acetylglucosaminyltransferase V activities in Rous sarcoma-transformed baby hamster kidney (RS-BHK) and BHK cells

Abstract

Recent studies have desmonstrated that Rous sarcoma virus‐transformed baby hamster kidney (RS‐BHK) cells express twofold higher levels of those N‐linked oligosac‐charides that contain the sequence [GlcNAc‐β(1,6)Man (1,6)] compared to nontrans‐formed 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:α‐D‐mannoside β(l,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, GlcNAcβ(1,2)Man α(1,6)Manβ‐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 [GlcNAcβ(1,6) Man] sequences in the RS‐BHK cells. The apparent K_m, values of the enzymes in RS‐BHK and BHK cell sonicates for UDP‐GIcNAc 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.