FIGURE 3.

Comparison of CS chain lengths polymerized using GalNAcβ1–4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-[³²P]phosphate)-TM as an acceptor substrate. The ³²P-labeled phosphorylated pentasaccharide linkage structure GalNAcβ1–4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-[³²P]phosphate)-TM was tested as an acceptor in polymerization reactions. The structure was co-expressed with the enzyme sources ChSy-1/ChPF (closed triangles), ChSy-1/ChSy-2 (open triangles), ChSy-1/ChSy-3 (closed circles), ChSy-2/ChPF (closed squares), ChSy-2/ChSy-3 (open squares), and ChSy-3/ChPF (open diamonds). ³²P-labeled polymerization reaction products were first isolated by gel filtration, subjected to reductive β-elimination using NaBH₄/NaOH, and then rechromatographed using a Superdex 200 column with 0.25 m NH₄HCO₃ and 7% 1-propanol as the eluent. Inset, the calibration curve denoting the linear relation between the log M_r and elution volume generated using the data obtained with commercial polysaccharides of known sizes (dextran average M_r, 200,000, 65,500, 37,500, and 18,100; all from Sigma). The numbered arrowheads 65.5, 37.5, and 18.1 indicate the eluted positions of the 65.5-, 37.5-, and 18.1-kDa saccharides, respectively. The total volume was a fraction of ∼60 (not shown).