Reactions were carried out in 0.1 M MES pH 6.5 with 10 mM MnCl2, 5 mM MgCl2, 40,000 DPM UDP-[14C]GlcA and 10 mM non-radioactive UDP-GlcA and UDP-Xyl each. Aliquots were removed at the displayed time points, boiled, and processed using RP C18 spin columns to separate untransferred donor from substrate. (A) The initial transfer of GlcA by LARGE to Xyl-α-pNP is slow in the absence of B4GAT1 but after transfer of the first GlcA polymerization rates increase to mirror those of LARGE in the presence of B4GAT1. (B) With Xyl-β-pNP as substrate, B4GAT1, which only possesses glucuronyltransferase activity, transfers a single GlcA per molecule of substrate. LARGE is unable to transfer GlcA to Xyl-β-pNP. Polymerization is only observed with the addition of both enzymes to the reaction mixture.