XENOBIOTIC GLUCOSYLTRANSFERASE ACTIVITY FROM SUSPENSION-CULTURED GLYCINE-MAX CELLS

Proteins extracted from suspension-cultured soybean (Glycine max (L.) Merr. Corsoy 79) cells contained O- and N-glucosyltransferases (GTs; EC 2.4.1) that catalyzed glucosylation of several xenobiotic compounds including (a) the hydroxylated herbicide metabolites 6-hydroxybentazone (B-6-OH), 8-hydroxybentazone and 5-hydroxydiclofop (b) the herbicide chloramben and (c) the environmental contaminants 2,4-dichlorophenol and 3,4-dichloroaniline. The O-GT that catalyzes B-6-OH glucosylation, UDP-glucose: B-6-OH glucosyltransferase (B6GT), was chosen for further study. A rapid and sensitive B6GT assay was developed that uses ethyl acetate extraction to separate the product 6-O-[C-14]glucosylbentazone from the glucose donor uridine[5']diphospho-[1]-alpha-D-[U-C-14]glucose. B6GT, recovered in the soluble protein fraction, was extracted in consistently high amounts from Corsoy 79 cells cultured for one to seven days. 2-Mercaptoethanol in the extraction buffer increased B6GT activity as did sodium, potassium, calcium, magnesium or manganese(II) chlorides in the assay buffer. B6GT activity in an ammonium sulfate fraction (30-50% saturation pellet) displayed two pH optima, one at pH 5.5 and another at pH 10 to 11. Apparent K-m values for UDP-glucose and B-6-OH in the ammonium sulfate fraction were 90 and 4.5 mu M, respectively. Thus, we have partially characterized glucosylation of B-6-OH, one example of the several xenobiotic GT activities present in Corsoy 79 soybean.