Figure 2. Release of hyaluronan from the membrane-bound synthase.

Hyaluronan was synthesized by B6 membranes in the presence of a substrate containing 8 μM UDP-[¹⁴C]GlcA and unlabelled 166 μM UDP-GlcNAc (■, □) or a substrate diluted 1:2 (●, ○) or 1:3 (▲, △) with a buffer (4 mM dithiothreitol and 20 mM MgCl₂ in 50 mM Tris-malonate). At the times indicated, the membranes were sedimented for 3 min at 14000 g, dissolved in 1% SDS and applied to descending paper chromatography (solid symbols). The supernatants (open symbols) were applied directly [1]. The origins containing hyaluronan were excised and the radioactivities were determined (A). From these data, the ratios of released hyaluronan and membrane-bound hyaluronan were calculated (B).