FIG. 2.

Glycolipid products are sensitive to saponification and PLD treatment. Lipids were labeled by incubating WU2 membranes in reactions containing 100 mM HEPES (pH 7.5), 10 mM MnCl₂, and either 1 μM UDP-[¹⁴C]Glc or 1 μM UDP-[¹⁴C]GlcUA for 20 to 30 min at 35°C. Membranes were sedimented and washed twice with 100 mM HEPES (pH 7.5) containing 10% glycerol and then solubilized with 0.5% Nonidet P-40 containing 0.05 M EDTA. Solubilized products were saponified with 0.1 N NaOH for 20 min at 35°C or treated with 5 units of PLD for 1 h at 30°C in 25 mM Tris-acetate (pH 8) containing 10 mM CaCl₂. (A) Untreated, saponified, and PLD-treated [¹⁴C]GlcUA-labeled samples were subjected to TLC on silica plates in butanol:acetic acid:water (44:16:40). Products were visualized by autoradiography. (B) Untreated (•), saponified (○), and PLD-treated (▴) [¹⁴C]Glc-labeled samples were chromatographed on paper in butanol:acetic acid:water (44:16:40) for 2 h. The chromatogram was cut into 1-cm strips, and the amount of radioactivity was determined by liquid scintillation counting.