Measuring asparagine synthetase activity in crude plant extracts

Asparagine synthetase B (AS) is the primary enzyme responsible for asparagine synthesis in plants. Routine biochemical studies of this enzyme's activity have been hindered by several problems including enzyme instability and rapid physiological turnover, endogenous inhibitors, competing pathways, and asparaginase activity. We describe an extraction procedure and assay conditions that provide a reliable, direct assay for the determination of AS activity in crude plant extracts. This assay performed well with several leguminous species and the enzyme preparation retained activity for up to 3 weeks when stored at -80 degrees C. Radio-HPLC detection enabled quantitative measurement of de novo aspargine synthesis in the extracts. Optimal activity was obtained with 1 mM glutamine and 10 mM ATP in the reaction assay. Aminooxyacetic acid (AOA, 1 mM) which prevents the assimilation of aspartate into the TCA cycle, was necessary to measure AS activity in peas, but not in lupine or soybean.