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. 2018 Oct 16;293(49):19047–19063. doi: 10.1074/jbc.RA118.004463

Figure 3.

Figure 3.

Elongation of medium-chain-length HG acceptors (DP11–15) by GAUT1:GAUT7. A, size-exclusion chromatography of the products synthesized in a reaction containing 100 nm GAUT1:GAUT7, 1 mm UDP-GalA, and 10 μm DP11 acceptor. Reactions were incubated for the indicated times and injected into a Superose 12 column. Selected peaks are indicated. Arrows, peak retention volume of dextran standards (270-, 150-, 50-, and 12-kDa average MW). B, high-percentage polyacrylamide gel stained with a combination of alcian blue/silver. Reactions containing a 10 μm concentration of a DP11 acceptor were incubated, and aliquots representing 200 ng of acceptor oligosaccharide were separated on a 30% polyacrylamide gel. HG standards include the HG oligosaccharide acceptor mix (lane 1) and high-MW commercial polygalacturonic acid (PGA) (lane 2). Individual bands of discrete DP that can be counted in the HG standard are indicated to the left of the gel. C, size-exclusion chromatography of products synthesized in a reaction containing 100 μm DP11 acceptor. D, high-percentage PAGE of a reaction containing 100 μm DP11 acceptor. E, MALDI-TOF MS analysis of products produced by GAUT1:GAUT7 in reactions containing 100 μm DP15 HG acceptor and 1 mm UDP-GalA. Reaction products were reducing-end-labeled with 2-AB after the indicated reaction times. The series of ions (m/z) with a mass separation of 176 Da is consistent with the sequential addition of GalA to the DP15 acceptor.