CHARACTERIZATION OF O-GLUCOSYLTRANSFERASES WITH ACTIVITIES TOWARD PHENOLIC SUBSTRATES IN ALFALFA

O-glucosyltransferases (OGTs, EC 2.4.1.-) from seedlings and suspensio n-cultured cells of alfalfa (Medicago sativa) with activities toward e ndogenously occurring flavonoid (quercetin) and isoflavonoid (medicarp in, coumestrol, formononetin) substrates have been characterized and p artially purified. All OGT activities were cytosolic, had a pH optimum of pH 9, were stimulated by thiol reagents, inhibited by a range of s alts and utilized both uridine diphosphate glucose (UDPG) and UDP-gala ctose as sugar donors. Activity toward endogenous substrates was in th e order quercetin > coumestrol > medicarpin > formononetin. K(m)s towa rd flavonoid/isoflavonoid substrates were in the range 12-57 and 13-20 mu M toward UDPG. Smaller enzyme activities were also observed toward related flavonoid and phenylpropanoid substrates. Specific OGT activi ties were greater in cell cultures than in plants. In seedlings the hi ghest activities toward flavonoids were observed in the foliage and to ward isoflavonoids in the roots. The OGT was extensively purified by a combination of size exclusion, hydrophobic interaction, chromatofocus ing and ion-exchange chromatography. During purification, evidence was obtained that the activities directed toward the flavonoid and pteroc arpan/isoflavonoid substrates were due to at least two closely related enzyme activities. All OGTs had M(r)s of 47 x 10(3), isoelectric poin ts between pH 4.6 and 4.7 and were co-eluted by anion-exchange chromat ography, but could be resolved as two forms by hydrophobic interaction chromatography. These OGTs are responsible for glucosylating a range of biologically active flavonoids involved in both the defence respons e and nodulation in alfalfa though no evidence was obtained that the e nzyme activities regulated the accumulation of conjugates.