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).