Figure 1. Purification of a Gal3ST from porcine colonic mucosa.

(A) Con A–Sepharose chromatography of the NaCl-eluted fraction of the second round of hydroxyapatite chromatography. The fractions were monitored for Gal3ST activity (●) and protein level (○). The arrow indicates the starting point of elution with 0.4 M α-methylmannoside in buffer C. Fractions indicated by the horizontal bar were collected. (B) Porcine colonic mucin-derived glycopeptide–Sepharose chromatography of NaCl-eluted fractions from the third round of hydroxyapatite chromatography. The fractions were monitored for Gal3ST activity (●) and protein (○). Fractions indicated by the horizontal bar were collected. (C) Cu²⁺-chelating Sepharose chromatography of the enzyme fractions obtained from mucin glycopeptide–Sepharose chromatography. The fractions were monitored for Gal3ST activity in the presence (●) or absence (○) of sphingosine. Fractions indicated by the horizontal bar were collected. (D) AMP–agarose chromatography of the enzyme fractions obtained from Cu²⁺-chelating Sepharose chromatography. The fractions were monitored for Gal3ST activity in the presence (●) or absence (○) of sphingosine. Fractions indicated by the horizontal bar were collected and used for the characterization of pGal3ST.