Figure 6.

Immunocomplexes from double transfectants efficiently convert GM3 to GM1. Truncated Gal-T2 affects the efficiency of conversion. To assess for the two-step conversion of GM3 to GM1, immunocomplexes from double (GalNAc-T/Gal-T2) or triple (GalNAc-T/Gal-T2/Gal-T2_1–52) transfectants or from mixtures of immunoprecipitates from single transfectants, as indicated, were incubated in a final volume of 20 μl with 100 μM UDP-GalNAc, 10 μM UDP-[³H]Gal (1 × 10⁶ cpm), 20 mM MnCl₂, 100 mM sodium cacodylate, pH 7.2, 3 mM CDP-Choline, 20 μg Triton CF-54/Tween 80 (2:1, wt/wt) for 2 h at 37°C in the presence or absence of 400 μM GM3 (see Fig. 1A). Reaction products were processed for lipid-bound radioactivity determination and HPTLC as indicated (4). Results are the mean ± SD of three independent experiments. (Inset). [³H]GM1 was identified as the main radioactive product of incubates with immunocomplexes of double (lane 1) or triple (lane 2) transfectants, after HPTLC of the lipid extracts and phosphorimaging. Lane 3 shows the positions of cochromatographed radioactive glycolipid standards obtained from clone 3 metabolically labeled from [³H]Gal.