The homogenate was centrifuged (10 min at _19 000 g) and the resulting supernatant was centrifuged again for 1 min at _19 000 g.
Proteins were determined according to Bradford (1976) and UFGalT activity was assayed by the method of Gerats et al. (1984).
The reaction mixture contained 40 to 130 ml of enzyme extract, 1 mM UDPGalactose in 20 ml
of a 330 mM solution of quercetin in methanol and additional extraction buffer to
produce 200 ml total volume. Samples were incubated for 15 min at 3008C.
Reaction was terminated by adding 800 ml of a 2 : 1 mixture of chloroform : -
methanol (with 1% HCl) and mixing.
The mixture was centrifuged at 15 000 g for 30 s, and in the biphasic partition the flavonoids were concentrated in the upper 400 ml phase.
Reaction products were separated on a high-performance liquid chromatograph equipped with a C18 column. Using an acetic acid : methanol : - water mixture (10 : 25 : 65) as eluting solvent, quercetin and its galactoside, quercetin-3-galactoside, were detected at 254 nm. The flow rate was 3 ml/min, which resulted in retention times of 2.3 min for quercetin-3-galactoside and
4.3 min for the aglycone.