1
262
N. Kovinich et al. / Phytochemistry 71 (2010) 1253–1263
removal of the supernatant and concentrated to 100
gas flow. All other reactions were stopped by vortexing in EtOAc
500 l) for 20 s followed by removal of the organic phase, which
was then evaporated under a stream of N . The residue was resus-
pended in HPLC grade MeOH (100 l). All reactions were filtered
through Teflon (0.2 m; Chromatographic Specialties) and 20
aliquots were analyzed by HPLC-DAD.
l
L under a N
2
by photospectroscopy using a NANODROP 2000 (Thermo Scien-
tific) the formula A530–0.25A657 to compensate for chlorophyll
absorption at 530 nm (Mancinelli, 1990).
(
l
2
l
Acknowledgments
l
ll
The authors would like to thank Vincenzo De Luca for the many
fruitful discussions and advice on this topic over the past couple of
years. We thank Kohei Kazuma and Naonobu Noda for providing
data on the Ct3GT-A substrate specificities. We also thank Ming
Hu for constructing the pCAMBIA2300-tCUP4::UGT78K1-tNOS
plasmid. We are grateful to the USDA for supplying the black
soybean line and the Arabidopsis Biological Resource Centre for
the T-DNA knockout line. We thank Susan Aitken for helpful
discussions on enzyme kinetics and Gregg Robideau for the
assistance with the phylogenetic analysis. This research was
funded under AAFC Research Branch Project Inventory Number
5.10. Enzyme kinetics
To measure the reaction velocity of the recombinant enzyme for
acceptor substrates, the concentrations of cyanidin (1) and
kaempferol (3) were varied from 3 to 100 M and from 5 to
00 M, respectively, and 5 mM UDP-glucose was used as donor
l
5
l
substrate. Enzyme assays were performed as described above but
reactions were stopped at 3 min and 5 min ensure linearity of data
points. Lineweaver–Burk graphs were plotted for the calculation of
m
K and Vmax values.
1
26 and by an NSERC Discovery Grant to JTA. NK was supported
through an NSERC Discovery Grant to BM.
5.11. Enzyme specificity
Appendix A. Supplementary data
The recombinant enzyme was tested for activity against antho-
cyaninidins (cyanidin (1), delphinidin, pelargonidin, petunidin,
peonidin, malvidin), anthocyanins (3-O-glucosides of the anthocy-
anidins and cyanidin 3-O-galactoside), flavonols (kaempferol
(
(
3), quercetin, myricetin, isorhamnetin), flavonol-3-glucosides
3-O-glucosides of kaempferol, quercetin, and isorhamnetin),
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