2.6 Assay for biuret hydrolase activity
Crude plant extracts were prepared as described below. Shoots of individual 19-day-old wild-type Nipponbare and transgenic B3-9-1 plants and 16-day-old B2-3-3-3 plants were weighed, and ground into powders under liquid nitrogen using a mortar and pestle. Then, the powdered tissue was homogenized in ten volumes of 25 mmol L-13-(N-morpholino) propanesulfonic acid (MOPS) buffer (pH 8.0). After centrifugation, the supernatant was used for the enzyme assay. Extractions were performed in duplicate for transgenic lines, and in triplicate for wild-type plants.
The biuret hydrolase assay was carried out as described by Martinez et al. (2001). The assay solution contained 50 mmol L-1sodium phosphate buffer (pH 8), 3 mmol L-1 biuret, and crude cell extracts. The assay mix was incubated at 30ÂșC, and the reaction was stopped by the addition of 0.5 mol L-1H2SO4. The amount of ammonia released from biuret was colorimetrically determined by indophenol blue method (Weatherburn, 1967). Protein concentrations were determined by the Bradford method, using Protein Assay CBB Solution (Nacalai tesque, Kyoto, Japan). Measurements were performed in duplicate.