Inhibition assay for allantoinase activity
Allantoinase activity was assayed according to Duran and Todd (2012).
Shoots of 9-day-old Nipponbare seedlings grown without biuret were
weighed and homogenized with a five-fold volume of extraction buffer
containing 50 mmol L-1 Tricine (pH 8.0) and 2 mmol
L-1 MnSO4. After centrifugation, the
supernatant was used in the inhibition assay. The enzymatic reaction was
initiated by the addition of allantoin as a substrate at a final
concentration of 10 mmol L-1 to the supernatant.
Biuret, 0, 0.5, and 5 mmol L-1 at final concentration,
were added to the reaction mixture together with the allantoin. The
mixture of 0.50 mL in a total was incubated at 30 ÂșC for 30 min, and the
reaction was stopped by adding 0.25 mL of 0.15 mol L-1HCl. Allantoic acid in the reaction mixture was colorimetrically
determined (Young & Conway, 1942). The allantoic acid content of the
crude extract was also determined and subtracted.
Protein concentrations in the crude extracts were determined by the
Bradford method using Protein Assay CBB Solution (Nacalai Tesque, Kyoto,
Japan).