Udp galactose

4-Hydroxy-2,5-dimethylfuran-3(2H)-one (HDMF), 2 (or 5)-ethyl-4-hydroxy-5 (or 2)-methylfuran-3(2H)-one (EHMF), 4-hydroxy-5-methylfuran-3(2H)-one (HMF), naringenin, eugenol, vanillic acid, salicylic acid, 1-naphthol, farnesol, sorbic acid, nerolidol, geraniol, fisetin, vanillin, chlorogenic acid, 2-naphthol, caffeic acid, methyl salicylate, ferulic acid, 1-octanol, and myricetin were purchased from Sigma-Aldrich (Shanghai, China). UDP-glucose, UDP-galactose, and UDP-glucuronic acid were purchased from Promega (Madison, USA). UDP-Xylose was purchased from Angfei Biotech Co., Ltd. (Guangzhou, China).

Enzyme activity measurements were conducted using the UDP-Glo Glycosyltransferase assay kit (Promega) according to the kit manual. Prior to the experiment, B4GalT1 was dialyzed against a buffer containing 20 mM Tris-HCl and 150 mM KCl at pH 7.0. The reaction buffer used for the assay was 50 mM Bis-Tris, 5 mM MnCl₂ pH 6.3. For the assay, a standard amount of 80 ng of B4GalT1 was used, with UDP-galactose (Promega) and ovalbumin (Sigma-Aldrich) as the donor and the acceptor, respectively. The donor was serially diluted 11 times with the highest concentration being 4 mM, while the acceptor concentration was kept constant at 3 mM. Measurements were done in triplicate after 1-hour incubation at 37°C. The reaction was stopped by the addition of the UDP detection reagent. The luminescence values of the samples were measured with a Tecan Infinite M1000 Pro luminometer.